Keywords: CRISPR
A distorter–restorer system drives quantitative reproductive isolation in rice
35461Zhang, Y., Yang, Y., Shi, C. et al., Nature Plants, 2026-02-24 10:08:29.
Hybrid sterility and segregation distortion are the major forms of postzygotic reproductive isolation in rice, yet the molecular basis of their quantitative variation remains unclear. Here we identify S44, a natural distorter–restorer system in Oryza longistaminata/Asian cultivated rice hybrids, comprising four tightly linked elements—Reproductive Isolation Distorter (RID), Reproductive Isolation Restorer (RIR), Reproductive Isolation Activator (RIA) and Reproductive Isolation Suppressor (RIS)—which collectively regulate hybrid male sterility and segregation distortion. The distorter RID triggers the elimination of O. sativa cultivar RD23 pollen, whereas the restorer RIR selectively safeguards O. longistaminata gametes, thereby preferentially transmitting its allele into the progeny. RIS and RIA fine-tune segregation distortion. We further demonstrate that the allelic conflicts at the S44 locus drive quantitative reproductive isolation between O. longistaminata and other rice lineages, and CRISPR-engineered RID knockout can universally overcome S44-mediated reproductive barriers in the AA genome, enabling revolutionary cross-species breeding. This distorter–restorer system provides a unique genetic module for deciphering speciation mechanisms and advancing crop breeding strategies.
Advances in CRISPR gene drives for mosquito population control
35449Robyn Raban, Anthony A James, Omar S Akbari, Current Opinion in Microbiology, 90. 2026-02-12 19:25:19.
CRISPR-based gene drive (GD) systems bias allele inheritance during meiosis, enabling transgenes to spread at rates exceeding Mendel’s law of segregation. This capability underlies their potential as powerful tools for controlling mosquito-borne diseases. GDs can be engineered either to suppress mosquito populations or to modify them by introducing traits that block pathogen transmission. Recent advances have focused on improving evolutionary stability, with modeling studies providing insights into expected population dynamics. With a focus on the most current population modification GDs, we discuss advances in GD architectures — including integral and allelic drives, combined modification–suppression systems, and both homing and non-homing toxin–antidote designs — that expand the range of possible strategies and address limitations of early homing drives. Numerous antipathogen effectors with strong pathogen-blocking activity can now be coupled to these systems, with current efforts assessing their durability against genetically diverse pathogens. Key challenges remain, including resistance evolution, ecological impacts, and long-term stability. Nonetheless, GDs offer a promising approach for reducing disease transmission, especially in regions where conventional interventions are difficult to sustain.
Assessing target genes for homing suppression gene drive
35447Xu, X., Fang, J., Chen, J. et al., The EMBO Journal, 2026-02-06 17:52:26.
Gene drives are engineered alleles that bias their own inheritance in offspring, enabling the spread of specific traits throughout a population. Targeting female fertility genes in a gene drive can be an efficient strategy for population suppression. In this study, we investigated nine female fertility genes in Drosophila melanogaster using CRISPR-based homing gene drives. Employing a multiplexed gRNA approach to prevent the formation of functional resistance alleles, we aimed to maintain high drive-conversion efficiency with low fitness costs in female drive-carriers. Drive efficiency was assessed in individual crosses and had varied performance across different target genes. Notably, drives targeting the octopamine β2 receptor (oct) and stall (stl) genes exhibited the highest drive-conversion rates and were further tested in cages. A drive targeting stl successfully suppressed a cage population with a high release frequency, though suppression failed in another replicate cage with a lower initial release frequency. Fitness costs in female drive carriers were observed in test cages, impacting the overall efficiency of population suppression. Further tests on the fertility of these lines using individual crosses indicated that some fitness costs were due to maternal deposition of Cas9 combined with new gRNA expression, which would only occur in progeny of drive males when testing split drives with separate Cas9 (when mimicking cages with complete drives) but not for complete drive systems. This could enable success in complete drives with higher maternal Cas9 deposition, even if cage experiments in split drives fail. Overall, our findings identify oct and stl as promising fertility targets and demonstrate both the potential and the constraints of fertility-based suppression drives, providing empirical evidence to guide the design and assessment of more efficient population control strategies.
Generating cisgenic sexing strains in insect pests
35435Davydova, S., Liu, J., Kandul, N.P. et al., Communications Biology, 2026-02-05 18:20:56.
Insect pest population control via sterile insect technique markedly benefits from separation by sex prior to release. To simplify this process, traditional genetics has been deployed to develop genetic sexing strains (GSSs) for several disease vectors and agricultural pests of vast economic significance, although very few are applied in the field due to associated fitness costs and instability. In this study, we generated a method to engineer cisgenic GSS (CGSS) in insects. We use CRISPR/Cas9-mediated homology-directed repair to seamlessly translocate a sex-specific alternatively spliced intron into a dominant phenotypic gene generating a genetically stable strain that enables sex-sorting by eye. To achieve this feat, we use Ceratitis capitata as our model and relied on the sex-specifically spliced intron of its endogenous transformer gene, which we seamlessly inserted a copy into the pupal colouration white pupae gene. This minimal modification resulted in the generation of a homozygous strain we term IMPERIAL that was genetically and phenotypically stable where all female pupae are brown while male pupae are white with overall good fitness. By minimally editing the genome, our novel CGSS approach can be applied to other pests that may aid more efficient and economically suitable pest control.
A conjugal gene drive-like system efficiently suppresses antibiotic resistance in a bacterial population
35426Kaduwal, S., Stuart, E.C., Auradkar, A. et al., npj Antimicrobials and Resistance, 4. 2026-02-05 10:30:00.
Antibiotic resistance (AR) is an escalating public health threat, necessitating innovative strategies to control resistant bacterial populations. One promising approach involves engineering genetic elements that can spread within microbial communities to eliminate AR genes. Previously, we developed Pro-Active Genetics (Pro-AG), a CRISPR-based gene-drive-like system capable of reducing AR colony-forming units (CFU) by approximately five logs. Here, we advance this technology by integrating Pro-AG into a conjugative transfer system, enabling efficient dissemination of an anti-AR gene cassette between two bacterial strains. Additionally, we characterize a complementary homology-based deletion (HBD) process, a CRISPR-driven mechanism that precisely removes target DNA sequences flanked by short direct repeats. Our findings reveal that Pro-AG and HBD are differentially influenced by the bacterial RecA pathway and that HBD components can be delivered via plasmids or phages to selectively delete Pro-AG cassettes. This built-in safeguard prevents uncontrolled spread of a gene cassette and mitigates unanticipated side effects. These refinements enhance the efficiency and flexibility of Pro-AG, expanding its potential applications in microbiome engineering, environmental remediation, and clinical interventions aimed at combating antibiotic resistance. More broadly, this work establishes a proof-of-principle for microbiome engineering strategies that could be leveraged to improve health and restore ecological balance.
Precision pest management: Genome editing tools, specifically CRISPR/Cas9 and future prospects
35424Ankush Saini, Neha Sharma, Nidhi Sharma, et al., Pesticide Biochemistry and Physiology, 218. 2026-02-03 15:42:17.
The growing resistance to synthetic insecticides and Bt toxins, alongside persistent crop losses despite heavy pesticide application, highlights the urgent need for safer, sustainable and efficient pest management strategies. This review presents genome editing as a precise and versatile approach to reduce pest impact by altering fertility, feeding patterns or vulnerability, while protecting beneficial organisms. Among the genome editing tools, CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9) is one of the most promising genome editing techniques in insects. It facilitates targeted functional studies, integration with RNAi and dual-expression systems and gene drive applications. Deployment is envisioned in two phases, initial laboratory modification followed by regulated field release, with a strong emphasis on biosafety through terminator genes, marked individuals for gene flow monitoring, optimized dosages, stringent screening and long-term ecological surveillance, along with transparency and adherence to international safety protocols. Significant challenges encompass delivery efficiency, identification of edits, off-target mutations, dose-related efficacy and sterility, unstable transmission and resistance development. Innovations such as base and prime editing minimize unintended mutations by circumventing double-stranded breaks (DSBs), while paratransgenic strategies targeting gut symbionts offer supplementary avenues; plant-mediated insect gene editing emerges as a promising frontier. Overall, carefully regulated trials aligned with policy frameworks and stakeholder involvement are vital to assess effectiveness in natural environments and achieve targeted, dependable and ecologically responsible pest control.
Targeted editing of pericentromeric satellite DNA alters sensitivity to meiotic drive
35388Danna G. Eickbush, Jabale Rahmat, Matthew Lindsay, et al., bioRxiv, 2026-01-21 16:51:02.
Eukaryotic genomes are abundant in satellite DNA (satDNA): large blocks of tandemly-repeated sequences that accumulate in heterochromatic genome regions. SatDNAs are dynamic in their genomic location and abundance across species. Some satDNAs overlap essential genome regions such as centromeres and telomeres, but even pericentromeric satDNA can have effects on phenotypes, raising questions about their functional significance. However, it remains unclear whether these effects depend on satDNA sequence, copy number, higher-order structural organization, or genomic context. The highly repetitive nature of satDNA arrays has long hindered detailed genomic and genetic analyses. Recent advances in long-read sequencing now facilitate both the detailed characterization of satDNA structure and the development of more targeted approaches to genetic analysis. Here we present a sequential CRISPR/Cas9-based strategy to make mutations in satDNA arrays and demonstrate its utility using an autosomal pericentromeric satDNA in Drosophila melanogaster called Responder (Rsp). Rsp is the target of a sperm-killing male meiotic driver, Segregation Distorter (SD), where sensitivity to sperm killing positively correlates with Rsp copy number. Using our CRISPR/Cas9 approach, we generated an allelic series of Rsp deletion and expansion variants in two genetic backgrounds and examined their effects on spermatogenesis. Our approach produced precise satDNA variants efficiently, with minimal detectable off-target effects. The resulting mutations affect sensitivity to SD that scale with Rsp copy number. This work establishes a new framework for experimentally dissecting satDNA function and provides insights into the evolutionary and functional roles of satDNA in genome organization.
Microhomology-mediated end joining is the predominant form of DNA repair in the mosquito Aedes aegypti with implications for gene editing, gene drive, and transgene removal
35375Joseph S Romanowski, Kevin M Myles, Zach N Adelman, Nucleic Acids Research, 54. 2026-01-20 16:30:25.
Programmable site-specific nucleases have revolutionized the field of genetics, and in the field of mosquito vector control, gene editing by these tools has inspired a new wave of population control approaches that aim to prevent disease transmission. Little is known of how DNA repair is prioritized in mosquitoes, which diverged from the nearest model system (Drosophila) by >200 million years, despite site-specific gene editing now being commonplace. Here, we report a scalable, high-throughput platform for studying DNA double-stranded DNA break (DSB) repair in mosquitoes by delivering CRISPR/Cas9, I-SceI, or other nucleases to Aedes aegypti embryos, capable of measuring single-strand annealing (SSA), non-homologous end joining, and microhomology-mediated end-joining (MMEJ) repair outcomes. We find CRISPR/Cas9 can induce deletions of up to 8.6 kb through SSA repair and is tolerant of resection distances of 3.5 kb. Indel events were insensitive to lig4 knockouts, and across 20 synthetic guide RNAs (sgRNAs) representing 5 locations in 2 transgenic strains were almost exclusively attributed to MMEJ repair, establishing MMEJ as the dominant form of repair in A. aegypti at CRISPR/Cas9 DSBs. This information is critical to our understanding of how DNA repair shapes processes required for genetic control strategies involving gene drive action/resistance as well as transgene stability.
Color-coded mosquitoes safely enables male-only releases to combat Dengue and Zika
35369Joshua Shavit, The Brighter Side of News, 2026-01-16 11:13:48.
Across much of the world, a tiny striped insect shapes whether families stay healthy or get sick. The Asian tiger mosquito carries Dengue, Zika and Chikungunya, and traditional control efforts often struggle to keep up. A new genetic trick that literally changes how these mosquitoes look could help tip the balance in your favor. Only female mosquitoes bite and pass on viruses. Males drink nectar, not blood. Many modern control programs release large numbers of males that are sterile or carry a trait that reduces survival in the next generation. When those males mate with wild females, fewer disease-carrying offspring survive. There is one big catch. These programs must release only males. If too many females slip through, they will still bite, still spread disease and may even weaken the program. Today, most facilities separate sexes by size during the pupal stage. That work is tedious, hard to automate and far from perfect. Researchers led by Doron Zaada and Prof. Philippos Papathanos at the Hebrew University of Jerusalem set out to remove that bottleneck. Their idea was simple and bold. Make male and female mosquitoes so visually different that machines, or even the human eye, can sort them at a glance. The team focused on Aedes albopictus, also known as the Asian tiger mosquito. It is aggressive, invasive and a major target for control programs worldwide. In their study, the scientists describe a “Genetic Sexing Strain” that turns sex into a visible trait. They used CRISPR gene editing to break a gene called yellow that controls dark pigment in the mosquito body. When this gene is disrupted, the insects turn pale, almost albino. The group then restored normal dark pigmentation only in males by linking a working copy of the yellow gene to nix, a sex-determining gene. Nix acts like a master switch. When it turns on in a mosquito, the insect develops as a fertile male, even if it started out genetically female. By tying yellow to nix, the team created a line in which all males are dark and all females remain pale. “This produces an engineered sex-linked trait in mosquitoes that uses the insect’s own genes,” said Prof. Papathanos. “By understanding and controlling the sex determination pathway, we were able to create a system were males and females are visually different at the genetic level.”
Scientists in Australia have created a genetically edited cane toad that gets stuck in the tadpole stage and attacks the plague before it spreads.
35365Noel Budeguer, Click Petróleo e Gás, 2026-01-16 10:59:37.
Australia has begun testing an unusual idea to combat one of the country's most persistent biological invasions: creating tadpoles of the cane toad who have never seen adults. The goal is to cut the problem off at the source, before the animals grow, leave the water, and move into new areas, increasing the impact on native wildlife. The proposal is noteworthy because it shifts the focus of control. Instead of targeting adults who have already spread out, the action targets the point where the population begins, the spawning grounds. The cane toad, a species Rhinella marina, it was introduced in Australia in 1935 and spread rapidly through northern regions. The species adapted quickly, encountered few natural barriers, and came to dominate environments where native animals cannot compete on an equal footing. Over time, the problem ceased to be isolated and became a constant threat to entire ecosystems. The adult stage is what allows the cane toad to move long distances and occupy new territories. The idea behind the project is to prevent this transition, keeping the animal confined to the aquatic environment and reducing the arrival of adults in the natural habitat. In practice, the plan attempts to halt the spread of the pest before it "jumps" out of the water. The technique uses CRISPR Cas9 to alter a point related to the hormonal control of metamorphosis. The target is the production of thyroxine, a hormone that triggers the transformation of the tadpole into an adult frog. Without this signaling, the animal remains in the aquatic phase and does not complete the cycle that would make it an even more aggressive terrestrial invader.
Research Advances and Challenges of Gene Drive Technology in Mosquito-Borne Disease Control
35363Yun Jiaqi, Ma Qin, Wang Guandong, et al., Laboratory Animal and Comparative Medicine, 45:773-783. 2026-01-14 12:01:39.
Mosquito-borne diseases (such as malaria, dengue fever, Zika virus disease, and Chikungunya) pose major threats to global public health, while traditional control methods based on chemical pesticides face severe challenges including enhanced drug resistance in vector mosquitoes and environmental pollution. Genetic control strategies have become high-potential alternative solutions for mosquito control due to their species specificity and environmental friendliness. Gene drive technology uses gene editing tools such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) to enable specific genes to efficiently spread in target mosquito populations through "super-Mendelian inheritance", offering a revolutionary strategy for the prevention and control of mosquito-borne diseases. This review systematically summarizes key advances, core challenges, and response strategies of gene drive technology in this field. Research advances: (1) In Anopheles malaria vectors, population suppression drives targeting sex determination genes or female reproductive genes can cause female sterility or skewed sex ratios to achieve population suppression. Population replacement gene drive strategies targeting host genes associated with Plasmodium infection or delivering anti-Plasmodium effector molecules in Anopheles can effectively block pathogen transmission. (2) In Aedes mosquito vectors of arboviruses, targeting female flight-essential genes achieves population suppression, and coupling of antiviral effector systems with drive elements is explored. Optimized split gene drive strategies demonstrate high cutting and recombination efficiency, and models predict safe and controllable spread of disease-resistance traits. (3) In Culex mosquitoes transmitting lymphatic filariasis, homology drive elements are integrated into two genes involved in the eye pigment synthesis pathway, allowing clear visualization of gene drive efficiency through eye color. Core Challenges: technological challenges include low homologous recombination repair efficiency, non-homologous end joining repair causing resistance allele generation, CRISPR/Cas9 off-target effects, and species adaptation differences. Ecological and safety challenges involve gene pool pollution caused by accidental spread of drive elements, potential ecological balance impacts, and long-term irreversible risks. Response strategies and prospects: employing multiplex guide RNA (gRNA) targeting strategies to enhance drive stability and combat potential resistance. Developing reversible designs such as synthetic resistance, reversal drives, and immunizing reversal drives as "genetic brakes". Establishing long-term ecological monitoring systems and mathematical modeling for risk assessment. Exploring "environmentally responsive drives" to enhance controllability. Future research should continuously optimize drive efficiency and specificity, deepen ecological risk evaluation, strengthen international cooperation, and promote ethical consensus and regulatory framework construction, with the aim of making gene drive technology a sustainable prevention and control strategy to address the global health challenge of mosquito-borne diseases under the premise of safety and controllability.
Behavior modification in fruit flies through male annihilation technique: field applications, olfactory mechanisms, and future directions
35329Liu, Wei; Zhang, Sijia; Wang, Guirong, Entomologia Generalis, 45:1565 - 1576. 2026-01-06 14:51:47.
The male annihilation technique (MAT), as a method of insect behavior manipulation, is an environmentally friendly approach that has been successfully applied in over 150 fruit fly eradication programs worldwide. Despite their effectiveness in integrated pest management programs, concerns have long persisted about the safety of the primarily used male lures – particularly regarding the potential toxicity of methyl eugenol (ME). In this review, we propose a novel attractant screening and validation system for the future improvement of male lures, based on recent advancements in functional genomics and genetic manipulation technologies achieved in tephritid fruit flies. This system is built upon our knowledge of tephritid fruit flies’ core olfactory signal transduction pathway (“Male lures → Odorant receptors → Odorant receptor neurons → Glomerulus”). The working pipeline is centered around a transgenic Bactrocera strain with labeled male lure-responsive olfactory receptor neurons (ORNs) as the key targets. Electrophysiological responses and calcium activity serve as the readouts, while the decision criteria focus on increased intensity, stability, and specificity of neural activation induced by candidate compounds. This pipeline, compared to traditional behavior-first methods, enables the efficient screening of computationally identified candidate compounds and provides a foundation for evaluating their field stability and safety. More importantly, it represents a shift from empirical, field-based optimization to a molecularly guided, receptor-based design framework, advancing the development of enhanced male lure solutions for future applications in insect behavior manipulation.
Researchers develop temperature-controlled gene-editing method to potentially improve efforts to control disease-carrying insects
35356Caliann Ferguson, UT Health Houston School of Public Health, 2026-01-06 09:54:29.
New research presents promising results from an innovative technique that utilizes temperature control to genetically engineer sterile populations of insects, such as mosquitoes responsible for diseases like malaria, dengue, and other vector-borne illnesses. Led by researchers at UTHealth Houston School of Public Health, the Nature Communications publication leverages historical and traditional sterilization insect techniques (SIT) and applies an innovative method that can be scaled for larger population protection. Used for decades, traditional SITs include releasing large numbers of sterile males into mosquito populations so that when they mate with wild females, no viable offspring are produced. CRISPR/Cas9-based methods have proven challenging as they require breeding two separate lines of engineered insects and carefully sorting those insects to produce and release only sterile males. Led by principal investigator Victor Lopez Del Amo, PhD, assistant professor of epidemiology, and Christina Nguyen, a research technician, who carried out most of the experiments, this promising method aims to simplify the traditional SIT by harnessing a gene-editing tool called CRISPR-Cas12a that can generate male sterility and female lethality in a temperature-controlled manner. Cas12a can be engineered to be inactive at lower temperatures and active at higher temperatures. This property enabled the team to develop a single genetically modified insect strain that possesses the genetic composition necessary to disrupt key fertility and reproductive genes.
Increasing the effective gene drive homing rate by targeting the haploinsufficient spermatogenesis gene KLHL10
35323Ceili L. Peng, W. Sebastian Kamau, Julien Freeman, et al., bioRxiv, 2026-01-05 12:04:02.
CRISPR-based gene drives represent a powerful new technology for limiting disease transmission and controlling invasive populations. These systems rely on homology-directed repair (HDR) to ‘drive’ a genetic element through a population. However, mammals tend to favor non-homologous end joining (NHEJ), which generates mutations that halt further drive propagation. Here, we describe the experimental characterization of a gene drive system targeting the haploinsufficient spermatogenesis gene KLHL10 in the laboratory mouse. Using a newly designed ‘coding sequence cassette’ we introduce downstream guide RNAs within the gene, ensuring that sperm undergoing NHEJ are selectively removed from the population. As a proof of principle, we demonstrate that targeting KLHL10 with constitutively expressed LbCas12a results in strong selection against frameshift-containing sperm, validating the core purification mechanism required for this drive strategy. Unexpectedly, we also observed that female offspring lacked most frameshift mutations, suggesting a previously unrecognized role for KLHL10 in oogenesis or early embryonic development.
New Gene Drive Stops the Spread of Malaria—Without Killing Any Mosquitoes
35343Shelly Fan, SingularityHub, 2025-12-18 11:51:00.
Mosquitoes are an uncomfortable, itchy nuisance. But for people in sub-Saharan Africa, a bite could mean death. The pests are living incubators for the parasite that causes malaria. Roughly 600,000 people are killed by the disease each year, with most being children under five years of age. Insecticides, malaria drugs, and mosquito nets saved a million lives globally in 2024 alone. But their efficacy is waning. Mosquitoes and the malaria parasite are becoming resistant to chemical inhibitors. And consistent, perfect use of physical barriers is hard to manage for years on end, especially for children. Realizing this, scientists have turned to a drastic solution: Gene drives, a technology that skews the rules of inheritance. Rather than nature’s fifty-fifty chance of an offspring inheriting a gene from either parent, gene drives raise the possibility of a gene’s inheritance to over 90 percent—if not higher. The tweak allows a gene to rapidly spread across entire populations. In lab tests encoding gene drives that reduce female mosquito fertility, mosquito populations have collapsed. Other experimental gene drives encoding genes that block parasite reproduction have suggested they could replace a natural population with one unable to carry malaria in just a few generations.
Strategies to improve the efficiency of homing gene drives with multiplexed gRNAs
35416Chen, W., Wu, P. & Champer, J., BMC Biol, 24. 2025-12-12 16:50:27.
CRISPR homing gene drive holds great potential for pest control, but its success is challenged by the generation of resistance alleles through end-joining repair. Using multiple gRNAs to target adjacent sites within a conserved gene can prevent functional resistance by allowing repeated cleavage events, but poor homology during DNA repair may compromise efficiency. We first assessed the efficiency of single gRNA drives with truncated homology arms in Drosophila melanogaster mimicking a multiplexed system in which only one site is cleaved. Integrating results into a detailed gRNA multiplexing model, we found that efficiency loss was greater than expected. To mitigate this, we evaluated two new strategies: (1) extended homology arms to span all target sites (with mutations in the PAMs to prevent self-cleavage) and (2) a population-level gRNA multiplexing system involving two or more drives, each carrying two gRNAs. Extended homology arms did not result in notable improvement in conversion efficiency, and the extended region could be lost during drive conversion. The population-level multiplexing gRNAs strategy was more promising, though the intentionally mutated PAM also could not be consistently inherited. Simulations of homing suppression drives applying population-level multiplexed gRNAs increased the success rate of population elimination and reduced the time required for suppression. Future drive designs requiring a larger number of gRNAs could potentially be improved. The design relying on extended homology arms may not represent an optimal strategy. However, population-level multiplexing gRNAs could serve as a promising alternative, enhancing efficiency while maintaining tolerance to functional resistance.
Genetic trick to make mosquitoes malaria resistant passes key test
35307Michael Le Page, New Scientist, 2025-12-10 17:06:06.
A genetic technology known as a gene drive could help prevent malaria by spreading genes in wild mosquitoes that stop them transmitting the parasite. Tests in a lab in Tanzania have now confirmed that one potential gene drive should achieve this if it were released in the country. “It would be a game-changing technology, that’s for sure,” says George Christophides at Imperial College London. A specific piece of DNA in the genome of an animal is normally passed on to only half its offspring, because a parent’s DNA is divided in half among egg or sperm. Gene drives increase this proportion, meaning a bit of DNA can spread rapidly through a population even if it provides no evolutionary benefit. There are many natural gene drives that work via all kinds of mechanisms – perhaps even in some human populations – and in 2013, biologists developed artificial gene drives using CRISPR gene-editing technology, which works by copying pieces of DNA from one chromosome to another. The idea is to use these drives to spread bits of DNA that block malaria transmission – but which bits? Christophides reported in 2022 that the development of malaria parasites inside mosquitoes can be greatly reduced by two tiny proteins, one derived from honeybees and the other from the African clawed frog.
Homing gene drive strains for genetic suppression of agricultural insect pests
35331Yadav, Amarish K.; Tarrand, Ariel E.; Scott, Maxwell J., Entomologia Generalis, 45:1577 - 1590. 2025-12-04 14:52:03.
Agricultural insect pests cause substantial losses in crop productivity each year. Genetic-based strategies provide economical and environmentally friendly ways to limit pests that reproduce sexually. In contrast to conventional genetic methods (e.g. SIT), homing gene drives (HGDs) are potentially capable of suppressing or modifying an entire pest population in a short period of time after releasing a small number of HGD insects. The advent of CRISPR/Cas gene editing tools has simplified the engineering of gene drives, and the progress made on HGDs in various insects in the recent past is encouraging. However, to date HGDs have been developed and evaluated in only a few agricultural pest species. These drives have been designed to suppress populations by targeting genes essential for female development or fertility. Homing gene drive relies on homology directed repair (HDR) of the Cas9-mediated double-stranded DNA break in germ cells. Consequently, the use of other DNA repair pathways such as non-homologous end joining (NHEJ) and micro-homology mediated end joining (MMEJ) can retard homing. Further, establishment of functional resistant alleles through these end-joining pathways is one of the major challenges associated with HGDs. Development of HGDs in some pest species is challenging due to the technical difficulties of making transgenics. Identification and characterization of germline-specific promoters and other regulatory elements to achieve precise HDR (in early meiosis) can facilitate efficient homing. In this review, we highlight the recent progress made towards developing HGDs in agricultural insect pests with insights gained from studies in model organisms (e.g. Drosophila melanogaster).
A temperature-sensitive CRISPR-Cas12a system for sterile insect technique
35361Nguyen, C., Omotayo, A.I., Sanz Juste, S. et al., Nature Communications, 16. 2025-11-14 11:49:30.
The sterile insect technique (SIT) reduces population numbers by releasing sterile males that produce non-viable progeny. Specifically, CRISPR/Cas9-based precision-guided SIT (pgSIT) generates sterile males through genetic crosses of two transgenic lines: a Cas9 strain and a guide RNA (gRNA) strain targeting male sterility and female viability or infertility. However, pgSIT requires separate maintenance of the two lines and sorting to obtain sterile males, creating possible challenges for scaling. To overcome this, we propose using Cas12a nuclease, which is inoperative at lower temperatures but active at higher temperatures. Here, we develop a Cas12a-based pgSIT system involving a single strain containing both the Cas12a nuclease and gRNAs to induce male sterility and female lethality. This strain can be maintained as a mixed stock of both sexes and only activated by increasing temperature, producing sterile males after just one generation. By reducing the challenges that arise with maintaining two separate lines, this system could offer a scalable alternative for vector control in combating vector-borne diseases.
Variants in Cas9 and nanos regulatory elements modulate activity and reduce resistance allele formation in homing gene drive
35294Ruizhi Zhou, Jie Du, Nicky R. Faber, Jackson Champer, bioRxiv, 2025-11-09 18:15:12.
Gene drive is a novel approach for controlling vector borne disease via either population modification or suppression. Even with high efficiency, though, overall drive performance can be reduced by somatic Cas9 expression and by maternal deposition of Cas9, leading to resistance allele formation. The nanos promoter for Cas9 shows very little leaky somatic expression, but it causes high rates of embryo resistance allele formation in Drosophila melanogaster. By truncating the promoter, we reduced rates of embryo resistance to undetectable levels, but germline cutting in females decreased by over half. Germline cutting and successful drive conversion was eventually lost when only the 5′ UTR was present, though males still retained moderate germline drive efficiency. Several additional methods were tested to improve performance, including additional suppressor elements to the 3′ UTR and introns to increase expression level. The most successful of these was the addition of a second nuclear localization signal, which substantially increased activity when coupled with a full-length or truncated nanos promoter. Overall, these experiments show the potential to modulate Cas9 regulatory elements to achieve desired expression for gene drive applications, while also showcasing the difficulty of obtaining an optimal activity profile.
Developing Y chromosome sex ratio distorters in the model insect Drosophila melanogaster
35259Yael Arien, Chen Zacharia, Elad Yonah, et al., bioRxiv, 2025-10-28 08:24:56.
CRISPR-Cas9 sex ratio distortion (SRD) systems can suppress insect populations by biasing progeny toward males, but realizing such systems requires reliable Cas9 expression from insect Y chromosomes. Here, we tested whether the spermatocyte-specific betaTub85D promoter can drive functional Cas9 expression when inserted on the Drosophila melanogaster Y chromosome. Using CRISPR-mediated homology-directed repair, we generated a Y-linked betaTub85D-Cas9-T2A-eGFP construct and compared its activity with an autosomal counterpart. Whereas autosomal betaTub85D-Cas9 induced strong male-biased sex ratios when paired with an X-poisoning gRNA, the Y-linked construct failed to distort sex ratios and exhibited approximately 2,000-fold reduction in Cas9 transcript abundance. Nonetheless, weak but detectable GFP fluorescence and Cas9 transcripts confirmed partial Y-linked promoter activity. These findings provide the first direct experimental evidence of meiotic sex chromosome inactivation (MSCI) acting on the Drosophila Y chromosome, revealing that meiotic promoters can remain weakly active despite strong repression. This work defines transcriptional limits of the Drosophila Y chromosome and informs the design of next-generation Y-linked gene drives for sustainable insect control.
Finding the perfect promoter: Correlating single-cell transcriptome data with gene drive performance
35228Yingke Wu, Yunchen Xia, Ziyin Yao, et al., bioRxiv, 2025-10-09 10:07:12.
Gene drive can control pathogen transmission or suppress vector populations by spreading drive alleles with super-Mendelian inheritance. CRISPR homing drive currently represents the most powerful type, and regulating Cas9 expression with specific promoters has been effective for improving drive performance. However, selecting these is often a major challenge. Here, we evaluated 35 Cas9 constructs driven by distinct promoters in different gene drive systems and identified associations between drive performance and single-cell RNA expression patterns of the promoter-associated genes. Our results indicate that higher drive conversion is significantly associated with elevated expression of the promoter-associated gene in the respective reproductive cells, but embryo resistance allele formation correlates with excessive female germline expression. For males, early germline expression produces superior performance. Thus, we find that optimal drive performance requires restricting Cas9 expression to a tight quantitative and spatiotemporal window. In addition, found that in situ integrated rhino-Cas9 constructs significantly reduce somatic expression, underscoring the importance of genomic locus. On the basis of these results, we propose criteria for selecting promoters, providing a theoretical rationale and practical guidance for optimization of promoter elements in homing gene drive systems.
CRISPR/Cas9-germline editing of Biomphalaria glabrata: A breakthrough in genetic modification of snails that transmit schistosomiasis
35226Kouhei Oonuma et al., Science Advances, 11. 2025-10-09 10:03:53.
Human schistosomiasis remains one of the most devastating parasitic diseases worldwide, and the development of genetically modified vector snails has long been a goal in the field. Here, we report the successful creation of genetically modified Biomphalaria glabrata, an important intermediate host, using CRISPR/Cas9 gene editing. We targeted the fibrinogen-related protein 3.1 (FREP3.1) gene, confirmed stable germline transmission of the mutated gene, and established two different homozygous FREP3.1-edited lines. Disruption of the FREP 3.1 gene did not alter snail susceptibility to Schistosoma mansoni infection, possibly due to a limited role of FREP3.1 in resistance or to functional redundancy and/or compensatory expression within the highly diverse FREP gene family. Our study demonstrates successful germline editing, effective ex ovo culture of decapsulated embryos, and the generation of viable, genetically modified B. glabrata snails, thereby establishing a foundation for future genetic strategies to control schistosomiasis.
Estimated cost and operational structure of pgSIT malaria vector control programs in selected West African countries
35203William A.C. Gendron, Robyn Raban, Agastya Mondal, et al., Scientific African, 29. 2025-09-30 15:35:38.
Malaria control has primarily been achieved through vector control, but current methods are insufficient to achieve elimination. Precision guided sterile insect technique (pgSIT) is a mosquito suppression technique that generates sterile male mosquitoes for mass release. Our previous studies showed that this intervention is expected to be highly cost-effective in a malaria endemic region of West Africa, but these estimates used only 15-31% capacity for sex sorting, which is the limiting production step and a primary cost. We, therefore, determined the most cost efficient facility size by calculating the cost per million Anopheles gambiae suppressed as the facility was scaled up to suppress more mosquitoes. We developed an optimized facility size per 9.2 million mosquitoes suppressed, which can be a framework for scaling and increases the cost effectiveness of this intervention. The development of this intervention can potentially interrupt malaria transmission, strengthen local public health institutions, create manufacturing capacity, provide local jobs, and enhance regional health security capabilities that are more resilient to disruptions in supply chains and malaria investment.
The role of the transformer gene in sex determination and its employment in CRISPR/Cas9-based homing gene drive in the global fruit pest Drosophila suzukii
35185Dan Deng, Xueying Yi, Wen Wen, Liuqing He, Wei Peng, Insect Biochemistry and Molecular Biology, 184. 2025-09-29 08:45:13.
Sex determination of Diptera is established by the cascade genes such as transformer (tra), though the primary signals for sex determination differ among different insects. Here, we report the isolation, expression and function of tra gene in an invasive pest, Drosophila suzukii, and study the potential use of the D. suzukii tra (Dstra) gene in CRISPR/Cas9-based homing gene drive for genetic-based pest management. The Dstra gene is highly conserved in structure and has a sex-specific transcript. To test the function of this gene in sex determination, Dstra dsRNA was injected into embryos. Almost all XX embryos developed into masculinized phenotypic male adults with intersex morphology. Abnormal ovaries were revealed in XX pseudomales upon dissection. Based on the necessary role of Dstra for female development, we developed and evaluated a homing gene drive that targets Dstra in D. suzukii. The drive component consisting of multiplex Dstra single guide RNAs and Cas9 with Dsvasa promoter was introduced into the Dstra locus. Abnormal development of both the external genitalia and gonads was observed in G0 and G1 chromosomal female adults that expressed the male-specific doublesex (dsx) transcript. Interestingly, knocking out Dstra led to significantly reduced fertility in adults of corresponding sex and moderate transmission rates of the DsRed gene (63.54 %) were observed. Our results not only confirm the conserved function of the Dstra gene in sex determination, but also highlight the potential of sex conversion-based suppression gene-drive strategy targeting the Dstra gene in controlling of D. suzukii populations.
The role of the transformer gene in sex determination and its employment in CRISPR/Cas9-based homing gene drive in the global fruit pest Drosophila suzukii
35172Dan Deng, Xueying Yi, Wen Wen, Liuqing He, Wei Peng, Insect Biochemistry and Molecular Biology, 184. 2025-09-26 14:04:36.
Sex determination of Diptera is established by the cascade genes such as transformer (tra), though the primary signals for sex determination differ among different insects. Here, we report the isolation, expression and function of tra gene in an invasive pest, Drosophila suzukii, and study the potential use of the D. suzukii tra (Dstra) gene in CRISPR/Cas9-based homing gene drive for genetic-based pest management. The Dstra gene is highly conserved in structure and has a sex-specific transcript. To test the function of this gene in sex determination, Dstra dsRNA was injected into embryos. Almost all XX embryos developed into masculinized phenotypic male adults with intersex morphology. Abnormal ovaries were revealed in XX pseudomales upon dissection. Based on the necessary role of Dstra for female development, we developed and evaluated a homing gene drive that targets Dstra in D. suzukii. The drive component consisting of multiplex Dstra single guide RNAs and Cas9 with Dsvasa promoter was introduced into the Dstra locus. Abnormal development of both the external genitalia and gonads was observed in G0 and G1 chromosomal female adults that expressed the male-specific doublesex (dsx) transcript. Interestingly, knocking out Dstra led to significantly reduced fertility in adults of corresponding sex and moderate transmission rates of the DsRed gene (63.54 %) were observed. Our results not only confirm the conserved function of the Dstra gene in sex determination, but also highlight the potential of sex conversion-based suppression gene-drive strategy targeting the Dstra gene in controlling of D. suzukii populations.
Reprogramming Sex for Vector Control: Maleness-Associated Transgenes in Aedes albopictus
35151Doron Shalom Yishai Zaada, Philippos Aris Papathanos, Eric Marois, Current Opinion in Insect Science, 2025-09-22 10:21:15.
Among other challenges, the world currently faces the expansion of pest insects such as the tiger mosquito Aedes albopictus, a growing threat to public health due to the pathogens it can transmit. Current control approaches based on insecticides or elimination of mosquito larval breeding sites are insufficient to suppress this highly invasive species. The discovery of Nix, a gene necessary and sufficient to determine the male sex in this mosquito, opens new prospects for genetic control strategies, in particular those based on transgenes that convert females into males, or that reduce female fitness. Such forms of genetic control could be effective on larger spatial and time scales compared to classical control approaches. This overview of current and emerging genetic control strategies targeting Aedes mosquitoes emphasizes the unique characteristics of Ae. albopictus, that make it particularly amenable to masculinization-based genetic control.
Experimental demonstration of daisy chain gene drive and modelling of daisy suppression systems
35147Jialiang Guo, Weizhe Chen, Jackson Champer, bioRxiv, 2025-09-22 10:06:58.
CRISPR-based gene drive can address ecological problems by biased inheritance coupled with an effector for either population modification of suppression. However, the potential risk of uncontrolled spread impedes some applications of gene drive. Daisy chain gene drives have received much attention as a potential approach to overcome this problem. They potentially allow the efficient spread of drive elements in a target population, but are ultimately self-limiting. This is achieved by splitting a normal gene drive system into multiple dependent drive elements, where each element can bias the inheritance of another, except one non-driving element. With the successive loss of each chain link, the spread of transgenic elements will slow down and eventually stop. Here, we use modelling to assess the population dynamics of daisy chain drives in both panmictic and continuous space models, focusing on suppression systems. We find that achieving population elimination through a single release of daisy chain gene drives is possible but difficult, with relatively high requirements for drive performance and release size. These effects are substantially amplified in spatial models. We also constructed two configurations of daisy chain gene drives in Drosophila melanogaster as a proof-of-principle. One is a rescue drive for population modification, and the other aims for population suppression by targeting a haplosufficient female fertility gene. These drives all functioned within expectations at moderate efficiency in individual crosses. However, the drive design failed to spread in cage populations because of higher than expected fitness costs. Overall, our study demonstrates that daisy chain systems may be promising candidates for both modification and suppression, but challenges remain in both construction and potential deployment in large regions.
Are we winning the war on cane toads?
35089Tom Gurn, Particle, 2025-08-28 19:10:42.
In 1935, a species known as the giant neotropical toad (Rhinella marina) was introduced to Australia. Scientists hoped these amphibians would control native cane beetles, but cane toads quickly colonised the country and had no discernible impact on beetle populations. Many different methods are being tested to remove these warty beasts, including a toad containment zone and turning the toads into sausages. Now, almost a century later, scientists believe they have finally stumbled across a potential solution to one of the worst invasive species ever. Will clever gene-editing techniques finally rid this continent of the dreaded cane toad? Professor Emeritus Rick Shine led the team behind the new idea, based on CRISPR gene-editing techniques. “We seem to have found a chink in the toads’ armour,” says Rick. “If we could only stop their metamorphosing, we could have these eternal tadpoles,” he says. Searching for collaborators to test this cunning plan, Rick happened across molecular biologist Dr Maciej Masielko, who was immediately struck by the beauty of the idea. “We know enough about the biochemistry of metamorphosis to know what we would need to do,” says Maciej. “Like using CRISPR-Cas9 to delete a part of the cane toad’s DNA.”
Conditional expression of Cas9 and dCas9 in Lucilia cuprina reveals dCas9-associated lethality
35027Alexis Kriete, Tatiana Basika, Rossina Novas, et al., bioRxiv, 2025-08-17 19:42:28.
Conditional sex transformation systems are promising tools in the fight against insect pests. In this study, we developed and tested CRISPR-based, tetracycline-repressible sex transformation strains in the Australian sheep blowfly, Lucilia cuprina. Two CRISPR effector molecules, Cas9 and dCas9, were employed to target the sex-determining gene transformer with the goal of turning female blowflies into males. The Cas9 version of the system induced robust knockout of a visual marker gene but failed to trigger sex transformation without external provision of transformer-targeting sgRNAs. Furthermore, we found that dCas9 expression was linked to several deleterious phenotypes, including developmental delays, reduced body weight, and death. Our study provides the first proof-of-concept conditional CRISPR systems in L. cuprina, and suggests that while dCas9 is toxic at high levels in this species, Cas9 is well-tolerated and may be able to induce sex transformation with minor modifications to the system.
CRISPR Mosquitoes That Can’t Bite
35031Science Techniz, 2025-08-09 19:49:44.
Scientists have used CRISPR gene editing to alter female mosquitoes so that their proboscis — the needle-like mouthpart used to pierce skin — develops like a male’s. The consequence is simple and profound: modified females can no longer pierce skin and therefore cannot take a blood meal or transmit human diseases like malaria and dengue. Researchers identified a gene involved in the developmental pathway that produces the female proboscis morphology. Using CRISPR-based edits, they altered that gene’s function so that genetically female mosquitoes develop a male-like mouthpart. Because males naturally do not bite (they feed on nectar), the modification removes the biting behavior without fundamentally disrupting other survival traits in lab tests. It’s important to stress that this is a high-level description intended to explain the concept, not a protocol or “how-to.” The work is complex, tightly regulated, and performed under strict laboratory and ethical oversight.
Genome Editing in Insect Pest Control: Importance, Strategies, and Future Implications
35011Ipsita Samal, Tanmaya Kumar Bhoi, Deepak Kumar Mahanta, Ansh Raj, J. Komal, Alagesan Keerthana, Vinod Kumar Dubey, Genome Editing for Pest Management, 2025-08-06 11:45:12.
Genome editing is a cutting-edge tool in biotechnology which brought about substantial changes in a variety of areas, including agriculture and pest control. As properly managing insect pests is critical for protecting food supplies and ensuring produce security, insect species frequently cause severe problems by destroying crop production, resulting in significant financial losses, food scarcity, and insufficiency. Innovative breakthroughs in pest management technology have lately resulted in the replacement of traditional chemical pesticide applications with environmentally safe and non-polluting interventions. Genome editing has the potential to transform pest control by providing precise and targeted therapies for insect pests that can be managed permanently. Researchers have discovered that genome editing methods have created new possibilities for improving food safety, agricultural productivity, and sustainable agriculture affected by pests. Scientists may now offset insect-related shortcomings with creative ways and strategies that ensure environmentally friendly and sustainable farming practices and environmental preservation, thanks to the application of genome editing tools. This chapter focuses on the use of these state-of-the-art technologies in genome editing tools such as ZFN, TALEN, and CRISPR/Cas9 enables us to assess the viability of insect control strategies, that provide significant promise for next-generation approaches to several major pest management problems and allow the insertion of precisely tailored modifications into the genetic composition of pests. To fully utilize the technology and specifically its implications for more efficient management of insect pests, study, analysis, and collaboration across many sectors are essential, which has been centralized in the current chapter.
Sequence mismatch between gene-drive and target-site flanking regions significantly impairs homing efficiency in Culex quinquefasciatus
35007Tim Harvey-Samuel, Rajdeep Kaur, Philip T Leftwich, et al., bioRxiv, 2025-08-04 11:03:26.
CRISPR/Cas9-based homing gene-drives (homing-drives) hold enormous potential as control tools for mosquito disease-vectors. These genomically-encoded technologies spread themselves through target populations by creating double-stranded DNA breaks on homologous chromosomes, into which the homing-drives are copied (homed). Homing is dependent on sequence homology between the genomic regions flanking the transgene insertion and the break site. Homing efficiency (i.e. copying rate) substantially impacts the power of these systems: less efficient homing-drives spread slower, have fewer applications and are more resistance-prone. Understanding what influences homing-drive efficiency is therefore vital to the successful use of these technologies. Here we report a novel mechanism by which a homing-drives efficiency can be significantly impaired by natural sequence variation within a population into which it is spreading. Using a kmo-targeting split homing-drive in the West Nile virus mosquito Culex quinquefasciatus, we found that target-site heterology (sequence mismatch between the genomic regions flanking the target cut-site and the homing-drive transgene) of less than 10% reduced homing efficiency by up to 54%. While substantial research effort has been dedicated to increasing homing-drive efficiency through optimisation of within-construct components, our results highlight that the real-world efficacy of these systems may in part depend on variation beyond these controllable factors.
CRISPR-GPT for agentic automation of gene-editing experiments
35077Qu, Y., Huang, K., Yin, M. et al., Nat. Biomed. Eng, 2025-07-30 12:34:29.
Performing effective gene-editing experiments requires a deep understanding of both the CRISPR technology and the biological system involved. Meanwhile, despite their versatility and promise, large language models (LLMs) often lack domain-specific knowledge and struggle to accurately solve biological design problems. We present CRISPR-GPT, an LLM agent system to automate and enhance CRISPR-based gene-editing design and data analysis. CRISPR-GPT leverages the reasoning capabilities of LLMs for complex task decomposition, decision-making and interactive human–artificial intelligence (AI) collaboration. This system incorporates domain expertise, retrieval techniques, external tools and a specialized LLM fine tuned with open-forum discussions among scientists. CRISPR-GPT assists users in selecting CRISPR systems, experiment planning, designing guide RNAs, choosing delivery methods, drafting protocols, designing assays and analysing data. We showcase the potential of CRISPR-GPT by knocking out four genes with CRISPR-Cas12a in a human lung adenocarcinoma cell line and epigenetically activating two genes using CRISPR-dCas9 in a human melanoma cell line. CRISPR-GPT enables fully AI-guided gene-editing experiment design and analysis across different modalities, validating its effectiveness as an AI co-pilot in genome engineering.
Genetic tweak in mosquitoes blocks malaria transmission without affecting insect health
34978University of California - San Diego, Phys.org, 2025-07-23 09:38:04.
Mosquitoes kill more people each year than any other animal. In 2023, the blood-sucking insects infected a reported 263 million people with malaria, leading to nearly 600,000 deaths, 80% of which were children. Recent efforts to block the transmission of malaria have been stalled because mosquitoes have adapted resistance to insecticides and the parasites within mosquitoes that cause malaria have become resistant to drugs. These setbacks have been amplified by the COVID-19 pandemic, which impeded ongoing anti-malarial efforts. Now, researchers at the University of California San Diego, Johns Hopkins University, UC Berkeley and the University of São Paulo have developed a new method that genetically blocks mosquitoes from transmitting malaria. Their work appears in Nature. Biologists Zhiqian Li and Ethan Bier from UC San Diego, along with Yuemei Dong and George Dimopoulos from Johns Hopkins University, created a CRISPR-based gene-editing system that changes a single molecule within mosquitoes, a minuscule but effective change that stops the malaria-parasite transmission process. Genetically altered mosquitoes are still able to bite those with malaria and acquire parasites from their blood, but the parasites can no longer be spread to other people. The new system is designed to genetically spread the malaria resistance trait until entire populations of the insects no longer transfer the disease-causing parasites. "Replacing a single amino acid in mosquitoes with another naturally occurring variant that prevents them from being infected with malarial parasites—and spreading that beneficial trait throughout a mosquito population—is a game-changer," said Bier, a professor in the UC San Diego Department of Cell and Developmental Biology (School of Biological Sciences). "It's hard to believe that this one tiny change has such a dramatic effect."
CRISPR technologies for the control and study of malaria-transmitting anopheline mosquitoes
34964Smidler, A.L., Akbari, O.S., Parasites Vectors, 18. 2025-07-08 13:44:43.
Malaria is one of the deadliest diseases on the planet, killing approximately 600,000 people annually, and is transmitted by the bite of an anopheline mosquito. Anophelines, and the diseases they transmit, have changed the course of history and the fate of nations, and their successful control promises to end the transmission of malaria. With the advent of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technologies, the study and control of these deadly pests have been revolutionized. As the release of genetically modified anophelines is being considered, here we outline the advances in CRISPR/Cas9 technologies and how they have revolutionized the study of anopheline basic biology and the development of innovative vector control strategies. We outline the major findings of CRISPR-based basic biological research into traits relevant for vector control including, but not limited to, olfaction, chemosensation, neurobiology, and reproduction. Further, we summarize the advancements in CRISPR-based innovative vector control strategies, such as the precision-guided sterile insect technique (pgSIT), inherited female elimination by genetically encoded nucleases to interrupt alleles (IFEGENIA), X-shredder, Y-linked editors, and gene drives. All in all, this review summarizes the basic biological and vector control research undertaken using CRISPR since its advent approximately a decade ago.
Experimental Test of Evolutionary Safety of a CRISPR-Cas9 Gene-Drive Element
34958Michael S Overton, Sean E Guy, Xingsen Chen, et al., bioRxiv, 2025-07-08 13:24:10.
CRISPR-Cas9 gene drives (CCGDs) are powerful tools for genetic control of wild populations, with applications from disease eradication to species conservation. However, Cas9 alone and in a complex with gRNA can cause double-stranded DNA breaks at off-target sites, which could increase the mutational load and lead to unintended loss-of-heterozygosity (LOH) events. These undesired effects raise potential concerns about the long-term evolutionary safety of CCGDs, but the magnitude of these effects is unknown. To measure how the presence of a CCGD or a Cas9 alone in the genome affects the rates of LOH events and de novo mutations, we carried out a mutation accumulation experiment in yeast Saccharomyces cerevisiae. We found no detectable effects on the genome-wide rates of mutations or LOH events. Our power calculations suggest that CCGD or Cas9 affect these rates by less than 30%, which is much less than natural variation for these traits in yeast. A more detailed examination shows that CCGD or Cas9 may alter the lengths and genomic distributions of LOH events, but the statistical support for these effects is weak. Thus, our results demonstrate that CCGDs impose at most a weak additional mutational burden in the yeast model. Although mutagenic effects of gene drives need to be further evaluated in other systems, our results add credence to the proposition that the evolutionary risks posed by well designed gene drives are likely acceptable.
Gene Drive Mosquitoes: Can We End Malaria?
34930The Curious Scholarette, YouTube, 2025-06-19 14:56:01.
What if we could genetically modify mosquitoes to stop malaria in its tracks? In this episode of The Curious Scholarette, we explore the groundbreaking world of gene-drive technology — a powerful tool that could eliminate malaria-carrying mosquitoes by altering their DNA. But with great power comes great ethical debate. 🔬 In this video, we break down: What gene drives are and how they work How scientists are using CRISPR to disrupt mosquito reproduction or malaria transmission Evidence from field trials and lab experiments The potential risks: ecological disruption, irreversible changes, and bioethical concerns Perspectives from global health experts, bioethicists, and community leaders 📍 Why it matters: Malaria kills over 600,000 people each year, mostly in sub-Saharan Africa. Could gene-drive mosquitoes be the silver bullet? Or are we playing with fire? 🧪 Sources Cited: WHO Malaria Report
Efficient CRISPR-Cas9-mediated genome editing of the cane toad (Rhinella marina)
34887Michael Clark, Alexander T. Funk, Alex Paporakis, et al., bioRxiv, 2025-06-02 19:02:13.
Invasive species inflict major ecological, economic, social, and cultural harm worldwide, highlighting the urgent need for innovative and effective control strategies. Genome editing offers exciting possibilities for creating highly targeted control methods for invasive species. Here, we demonstrate CRISPR-Cas9 genome editing in the cane toad (Rhinella marina), one of Australia’s most notorious invasive species, by targeting the tyrosinase gene to produce albino phenotypes that provide clear visual markers for assessing editing efficiency. Microinjection of Cas9 protein and guide RNAs into one-cell zygotes resulted in 87.6% of mosaic larvae displaying nearly complete albinism, with 2.3% exhibiting complete albinism. For completely albino individuals, genomic analysis confirmed predominantly frameshift mutations or large deletions at the target site, with no wild-type alleles detected. Germline transmission rates reflected the extent of albinism in the mosaic adult, where we achieved maternal germline transmission rates of almost 100%. This technology, representing the first application of CRISPR-Cas9 in the Bufonidae family, opens possibilities for exploring both basic research questions and strategies for population control.
Optimization of SgRNA expression with RNA pol III regulatory elements in Anopheles stephensi
34801Gonzalez, E., Anderson, M.A.E., Ang, J.X.D. et al., Scientific Reports, 15. 2025-04-23 11:08:06.
Anopheles stephensi, a major Asian malaria vector, is invading Africa and has been implicated in recent outbreaks of urban malaria. Control of this species is key to eliminating malaria in Africa. Genetic control strategies, and CRISPR/Cas9-based gene drives are emerging as promising species-specific, environmentally friendly, scalable, affordable methods for pest control. To implement these strategies, a key parameter to optimize for high efficiency is the spatiotemporal control of Cas9 and the gRNA. Here, we assessed the ability of four RNA Pol III promoters to bias the inheritance of a gene drive element inserted into the cd gene of An. stephensi. We determined the homing efficiency and examined eye phenotype as a proxy for non-homologous end joining (NHEJ) events in somatic tissue. We found all four promoters to be active, with mean inheritance rates up to 99.8%. We found a strong effect of the Cas9-bearing grandparent (grandparent genotype), likely due to maternally deposited Cas9.
Stronger population suppression by gene drive targeting doublesex from dominant female-sterile resistance alleles
34799Weizhe Chen, Ziye Wang, Jackson Champer, bioRxiv, 2025-04-23 11:00:18.
CRISPR homing drives can be used to suppress a population by targeting female fertility genes. They convert wild-type alleles to drive alleles in the germline of drive heterozygotes by homology-directed repair after DNA cleavage. However, resistance alleles produced by end-joining pose a great threat to homing drive. They prevent further recognition by Cas9, and therefore weaken suppressive power, or even stop suppression if they preserve the function of the target gene. We used multiplexed gRNAs targeting doublesex in Drosophila to avoid functional resistance and create resistance alleles that were dominant female-sterile. This occurred because the male dsx transcript was generated in females by disruption of the female-specific splicing acceptor site. We rescued dominant sterility of the drive by providing an alternate splicing site. As desired, the drive was recessive female sterile and yielded high drive inheritance among the progeny of both male and female drive heterozygotes. The dominant-sterile resistance alleles enabled stronger suppression in computational models, even in the face of modest drive efficiency and fitness costs. However, we found that male drive homozygotes were also sterile because they used the rescue splice site. Attempts to rescue males with alternate expression arrangements were not successful, though some male homozygotes had less severe intersex phenotypes. Though this negatively impacted the drive, models showed that it still had significantly improved suppressive power. Therefore, this design may have wide applicability to dsx-based suppression gene drives in a variety of organisms with intermediate homing drive performance.
Mathematically modelling the population dynamics of CRISPR gene drive systems in the pine pest Sirex noctilio
34779Strydom, H., Ouifki, R., Chapwanya, M., bioRxiv, 2025-04-18 10:34:17.
Sirex noctilio is an invasive pest of pine that has caused significant economic damage in South Africa and many other Southern Hemisphere countries. Current management tools are not efficient in all cases and consequently there is a need for more efficient and targeted control measures. An emerging tool for pest management is the use of gene editing and associated gene drive systems. In this study, we aim to investigate the use of CRISPR-Cas gene drive systems in the management of S. noctilio in South Africa. As a first step, we developed a model for the population dynamics of S. noctilio, using historical national population monitoring data and incorporating the influence of two main biological control agents of the pest. We then modelled the influence of two different CRISPR-Cas systems on the population dynamics of S. noctilio namely, a baseline CRISPR model and Complementary Sex Determination CRISPR (CSD) model. Each model is used to simulate a male and female only introduction strategy to estimate the effectiveness of different methods of introducing the gene drive system. The model calibration was achieved by optimizing the model fit to existing data using the least squares technique. Results suggest that both CRISPR gene drive systems would be effective at controlling the population growth of S. noctilio at high levels of introduction, but overall population control would be hindered by practical limitations. Although only two CRISPR models were explored, the underlying population model serves as a framework for further studies into the population dynamics of Sirex noctilio, as well as many other CRISPR-Cas gene drive systems.
Mosquito Sex Separation using Complementation of Selectable Traits and Engineered Neo-Sex Chromosomes
34736Zaada, D.S.Y., Toren, O., Krsticevic, F., et al., bioRxiv, 2025-04-03 17:26:54.
Effective and scalable sex separation remains a critical challenge for mosquito genetic control strategies. Genetic sexing strains (GSS) address this by genetically linking maleness with selectable traits, enabling efficient removal of females before release. Here, we describe a robust platform for the development of GSSs in the invasive Aedes albopictus mosquito by integrating a CRISPR-engineered selectable phenotype with sex conversion via nix, the male-determining factor. As a proof-of-concept, we disrupt the yellow gene to generate a vivid pigmentation marker, then rescue its function in males using nix-containing transgenes, creating a stable strain where all females are yellow and all engineered males are dark. The resulting GSS males are fertile, robust, and despite lacking the ancestral M locus, exhibit gene expression profiles closely resembling wild-type males. We benchmark sex separation based on pigmentation and discover that yellow mutant females exhibit slower larval development, enhancing protandry-based sorting. The GSS strain is compatible with existing size-based sex sorting systems, allowing for improved separation accuracy through the integration of natural and engineered sexually dimorphic traits. Additionally, we find that GSS females lay desiccation-sensitive eggs, reducing the risk of accidental female releases. Our approach is the first to engineer a sex-linked selectable trait by precisely targeting an endogenous gene and restoring its function in males, establishing a versatile platform for GSS development in Aedes mosquitoes.
Challenges in developing a split drive targeting dsx for the genetic control of the invasive malaria vector Anopheles stephensi
34659Larrosa-Godall, M., Ang, J.X.D., Leftwich, P.T. et al., Parasites & Vectors, 18. 2025-03-25 09:18:22.
Anopheles stephensi is a competent malaria vector mainly present in southern Asia and the Arabian Peninsula. Since 2012, it has invaded several countries of eastern Africa, creating an emerging risk of urban transmission. Urgent efforts are required to develop novel and more efficient strategies for targeted vector control. CRISPR/Cas9-based homing gene drives have been proposed as attractive alternative strategies. Gene drives have the potential to spread a desired trait through a population at higher rates than via normal Mendelian inheritance, even in the presence of a fitness cost. Several target genes have been suggested and tested in different mosquito vector species such as Anopheles gambiae and Aedes aegypti. Several promising suppression drives have been developed in An. gambiae that target the sex determination gene doublesex (dsx). In this study, a geographically confineable gene drive system targeting dsx was developed (dsxgRNA). Here, a transgenic line which expresses Cas9 under the control of the endogenous zpg promoter was generated. Separately a transgenic line which expresses a gRNA targeting the female specific exon of dsx was inserted into that same target site. The reproductive fitness of males and females heterozygous and homozygous for this element was determined. A series of experimental crosses was performed to combine the two elements and assess the homing rate of the dsx element in a split drive system. The drive was able to home in a super-Mendelian rate comparable to those obtained by an autonomous drive in this species. Although inheritance rates as high as 99.8% were observed, potentially providing very potent gene drive, dominant effects on male and female fertility were observed, which would be sufficient to hinder spread of such a drive. Molecular analysis indicated that the gRNA expressing insertion disrupted normal splicing of dsx. These results should be considered when proposing the viability of dsx as a target gene for a population suppression gene drives in Anopheles stephensi. Although high homing rates were observed, the fitness defects found in both males and females carrying the transgene would likely prohibit this drive from functioning in the field.
Engineering drive–selection balance for localized population suppression with neutral dynamics
34597Willis, K., and Burt, A., Proceedings of the National Academy of Sciences, 122. 2025-03-18 10:29:28.
While the release of sterile males has been highly successful in suppressing some pest populations, it is impractical for many species due to the males disappearing after a single generation, necessitating large, repeated releases to maintain sufficient impact. Synthetic gene drives promise more efficient approaches since they can increase in frequency from rare, yet this also allows them to spread across a landscape, which may not always be desired. Between these two extremes are selectively neutral genetic constructs which persist at the frequency they are released, offering the potential for efficient suppression that remains localized. One way to achieve this would be to have perfect balance, at all construct frequencies, between gene drive increasing frequency and selection decreasing it. Here, we describe a way to closely approximate this balance using a toxin–antidote genetic construct that causes recessive lethality or sterility, encodes a genomic editor that makes dominant lethal or sterile edits in the genome, and provides protection against the action or consequences of the editing. Computer modeling shows that this design can be 100-fold more efficient than sterile males, increasing to 1,000-fold when released alongside a genetic booster. We describe designs for CRISPR-based molecular construction, including options that avoid using recoded genes as antidotes.
CRISPR/Cas9 Genome Editing in the Diamondback Moth: Current Progress, Challenges, and Prospects
34556Asad, M., Chang, Y., Liao, J., & Yang, G., International Journal of Molecular Sciences, 26:1515. 2025-03-04 11:34:07.
The development of site-specific genome-editing tools like CRISPR (clustered regularly interspaced short palindromic repeat) and its associated protein, Cas9, is revolutionizing genetic engineering with its highly efficient mechanism, offering the potential for effective pest management. Recently, CRISPR/Cas9 gene-editing has been extensively utilized in the management of the diamondback moth, Plutella xylostella (L.), a highly destructive pest of vegetable crops, for different purposes, such as gene function analysis and genetic control. However, the progress related to this gene-editing tool in P. xylostella has not yet been summarized. This review highlights the progress and applications of CRISPR/Cas9 in uncovering the genes critical for development, reproduction, and insecticide resistance in P. xylostella. Moreover, the progress related to the CRISPR/Cas9 gene drive for population suppression and modifications has also been discussed. In addition to the significant progress made, challenges such as low germline editing efficiency and limited homology-directed repair remain obstacles to its widespread application. To address these limitations, we have discussed the different strategies that are anticipated to improve the efficiency of CRISPR/Cas9, paving the way to it becoming a pivotal tool in sustainable pest management. Therefore, the present review will help researchers in the future enhance the efficiency of the CRISPR/Cas9 system and use it to manage the diamondback moth.
Engineering gene drive docking sites in a haplolethal locus in Anopheles gambiae
34552Andrea L Smidler, Eryney A Marrogi, Sean Scot, et al., bioRxiv, 2025-03-04 11:03:46.
Gene drives are selfish genetic elements which promise to be powerful tools in the fight against vector-borne diseases such as malaria. We previously proposed population replacement gene drives designed to better withstand the evolution of resistance by homing through haplolethal loci. Because most mutations in the wild-type allele that would otherwise confer resistance are lethal, only successful drive homing permits the cell to survive. Here we outline the development and characterization of two ΦC31-Recombination mediated cassette exchange (RMCE) gene drive docking lines with these features in Anopheles gambiae, a first step towards construction of robust gene drives in this important malaria vector. We outline adaption of the technique HACK (Homology Assisted CRISPR knockin) to knock-in two docking site sequences into a paired haplolethal-haplosufficient (Ribosome-Proteasome) locus, and confirm that these docking lines permit insertion of drive-relevant transgenes. We report the first anopheline proteasome knockouts, and identify ribosome mutants that reveal a major hurdle that such designs must overcome to develop robust drives in the future. Although we do not achieve drive, this work provides a new tool for constructing future evolution-robust drive systems and reveals critical challenges that must be overcome for future development of gene drives designed to target haplolethal loci in anophelines and, potentially, other metazoans.
A Gene Drive with a Disappearing Act Can Aid Pest Control
34537Shelby Bradford, PhD, The Scientist, 2025-02-21 12:02:41.
Geneticists developed a gene drive that reverts insecticide-resistant mutations in insects, using a system that gradually eliminates itself from the genome. This breakthrough offers a potential solution to combat insecticide resistance without permanently altering species, and could be adapted to mosquitoes and other pests in the future.
Controlling the frequency dynamics of homing gene drives for intermediate outcomes
34494Camm B., Fournier-Level A., G3: Genes|Genomes|Genetics, 2025-02-04 17:53:42.
Gene drives have enormous potential for solving biological issues by forcing the spread of desired alleles through populations. However, to safeguard from the potentially irreversible consequences on natural populations, gene drives with intermediate outcomes that neither fixate nor get removed from the population are of outstanding interest. To elucidate the conditions leading to intermediate gene drive outcomes, a stochastic, individual allele-focused gene drive model was developed to simulate the diffusion of a homing gene drive in a population. The frequencies of multiple alleles at a locus targeted by a gene drive were tracked under various scenarios. These explored the effect of gene drive conversion efficiency, strength and frequency of resistance alleles, dominance and strength of a fitness cost for the gene drive, and the level of inbreeding. Four outcomes were consistently observed: fixation, loss, temporary, and equilibrium. The latter 2 are defined by the frequency of the gene drive peaking then crashing or plateauing, respectively. No single variable determined the outcome of a drive. The difference between the conversion efficiency and resistance level, modeled quantitatively, differentiated the temporary and equilibrium outcomes. The frequency dynamics of the gene drive within outcomes varied extensively, with different variables driving these dynamics between outcomes. These simulation results highlight the possibility of fine-tuning gene drive outcomes and frequency dynamics. To that end, we provide a web application implementing our model, which will guide the safer design of gene drives able to achieve a range of controllable outcomes tailored to population management needs.
Establishment of pupal color as a screening marker and activity analysis of six U6 promoters in Zeugodacus cucurbitae using the white pupae gene
34487Fan Z., Wu Y., Zha X., Ma S., et al., Pest Management Science, 2025-02-04 17:36:39.
The genetic control method, which is environmentally friendly and species-specific, has effectively reduced or eliminated pests in many areas. One essential requirement to control a species is the identification of its genetic and molecular elements. Such elements, however, are rarely available in Zeugodacus cucurbitae, a very destructive insect pest worldwide. In this study, we knocked out the white pupae (wp) gene in Z. cucurbitae and generated a wp(−) strain, which has a white pupae phenotype. The white puparium color was successfully restored to brown by inserting the wp gene rescue allele into the genome of the wp(−) strain using piggyBac transgenic technology. The potential wp promoter was then truncated to drive the expression of the wp gene and the puparium color was rescued even by the 605 bp sequence upstream of its transcription initiation site. Further fertility tests showed that knocking out or rescuing the wp gene had no effect on the reproduction of adult flies. In addition, we identified six U6 promoters and tested their promoter activities in the embryos of Z. cucurbitae. The ZcU6-2 and ZcU6-1 promoters exhibited significantly higher activity than the others and are suitable for use in CRISPR technology-based genetic control methods. Our work first shows the success of applying piggyBac transgenic technology in Z. cucurbitae. Our results demonstrate a highly efficient transgenic screening marker by puparium color and the promoter activity of multiple ZcU6 promoters, facilitating the construction of transgenic strains that are used for genetic control of tephritid species. © 2025 Society of Chemical Industry.
Integrating malaria vaccine and CRISPR/Cas9 gene drive: a comprehensive strategy for accelerated malaria eradication
34374Abraham, I.C., Aboje, J.E., Ukoaka, B.M. et al., Malaria Journal, 24. 2025-01-28 15:04:09.
Malaria remains a significant public health challenge, particularly in low- and middle-income countries, despite ongoing efforts to eradicate the disease. Recent advancements, including the rollout of malaria vaccines, such as RTS,S/AS01 and R21/Matrix-M™, offer new avenues for prevention. However, the rise of resistance to anti-malarial medications necessitates innovative strategies. This review explores the potential integration of CRISPR/Cas9 gene drive technology with malaria vaccination efforts to enhance vector control and reduce transmission. By employing gene drive mechanisms for population suppression and replacement of malaria-transmitting Anopheles mosquitoes, combined with the immunogenic properties of vaccines, a synergistic approach can be established. This paper discussed the need for integrated strategies to address the biological complexities of malaria and socio-economic factors influencing its prevalence. Challenges such as regulatory hurdles, community acceptance, ecological impacts, and sustainable funding are examined, alongside strategies for implementation within existing malaria control programmes. This integrated approach could significantly contribute to achieving the World Health Organization's targets for malaria reduction by 2030, ultimately enhancing public health outcomes and supporting broader socio-economic development.
Gene drive-based population suppression in the malaria vector Anopheles stephensi
34372Xu, X., Chen, J., Wang, Y. et al., Nature Communications, 16:1007. 2025-01-28 14:41:08.
Gene drives are alleles that can bias the inheritance of specific traits in target populations for the purpose of modification or suppression. Here, we construct a homing suppression drive in the major urban malaria vector Anopheles stephensi targeting the female-specific exon of doublesex, incorporating two gRNAs and a nanos-Cas9 to reduce functional resistance and improve female heterozygote fitness. Our results show that the drive was recessive sterile in both females and males, with various intersex phenotypes in drive homozygotes. Both male and female drive heterozygotes show only moderate drive conversion, indicating that the nanos promoter has lower activity in A. stephensi than in Anopheles gambiae. By amplicon sequencing, we detect a very low level of resistance allele formation. Combination of the homing suppression drive and a vasa-Cas9 line boosts the drive conversion rate of the homing drive to 100%, suggesting the use of similar systems for population suppression in a continuous release strategy with a lower release rate than SIT or fsRIDL techniques. This study contributes valuable insights to the development of more efficient and environmentally friendly pest control tools aimed at disrupting disease transmission.
Gene Drive Technology Offers Hope For Malaria Vector Control
34365Evrim Ağacı, The Pinnacle Gazette, 2025-01-28 13:46:11.
A team of scientists has developed an innovative gene drive targeting Anopheles stephensi mosquitoes, a key malaria vector in urban areas. Their strategy uses CRISPR technology to disrupt the doublesex (dsx) gene, crucial for female fertility, thereby reducing mosquito populations. The gene drive, called HSDdsx, showed promising results in suppressing mosquito reproduction with minimal resistance, indicating long-term effectiveness. This approach could help control malaria transmission and reduce reliance on pesticides. The research also opens doors for future advancements in pest control, with potential applications against other disease vectors, marking a significant step toward environmentally friendly solutions.
Assessment of drive efficiency and resistance allele formation of a homing gene drive in the mosquito Aedes aegypti
34261Yang, X., Xu, X., Chen, Y. et al., Journal of Pest Science, 2025-01-14 09:57:33.
Aedes aegypti, known for transmitting viruses such as dengue, Zika and yellow fever, poses a significant public health threat. Conventional insecticides give rise to a range of issues, including ecological contamination and insect resistance. Hence, there is a pressing demand for environmentally-friendly, safer and more efficacious strategies for mosquito control. With the rapid advancement of the CRISPR/Cas9 system in gene function exploration and pest population control, substantial progress has been achieved in utilizing CRISPR/Cas9-based gene drive systems across various mosquito species. Only a few studies on gene drive technology have been conducted in Ae. aegypti. In this study, we constructed two complete drives for Ae. aegypti with different Cas9 promoters, each targeting kmo. Our drive based on PubCas9 had limited activity, but one with ExuCas9 exhibited super-Mendelian inheritance rates of approximately 60%. We observed low but detectable somatic activity of the drive and no evidence of maternally deposited Cas9. Germline resistance allele formation rates were similar to drive conversion rates, but most wild-type alleles in the germline remained uncut. Injections into the ExuCas9 drive line had 100% knockout efficiency among surviving offspring at three separate target genes. These results support the development and application of novel genetic pest control technologies aimed at combating Ae. aegypti.
Identification of novel genes responsible for a pollen killer present in local natural populations of Arabidopsis thaliana
34259Ricou, A., Simon, M., Duflos, R., et al, PLOS Genetics, 21. 2025-01-14 09:53:12.
Gamete killers are genetic loci that distort segregation in the progeny of hybrids because the killer allele promotes the elimination of the gametes that carry the sensitive allele. They are widely distributed in eukaryotes and are important for understanding genome evolution and speciation. We had previously identified a pollen killer in hybrids between two distant natural accessions of Arabidopsis thaliana. This pollen killer involves three genetically linked genes, and we previously reported the identification of the gene encoding the antidote that protects pollen grains from the killer activity. In this study, we identified the two other genes of the pollen killer by using CRISPR-Cas9 induced mutants. These two genes are necessary for the killer activity that we demonstrated to be specific to pollen. The cellular localization of the pollen killer encoded proteins suggests that the pollen killer activity involves the mitochondria. Sequence analyses reveal predicted domains from the same families in the killer proteins. In addition, the C-terminal half of one of the killer proteins is identical to the antidote, and one amino acid, crucial for the antidote activity, is also essential for the killer function. Investigating more than 700 worldwide accessions of A. thaliana, we confirmed that the locus is subject to important structural rearrangements and copy number variation. By exploiting available de novo genomic sequences, we propose a scenario for the emergence of this pollen killer in A. thaliana. Furthermore, we report the co-occurrence and behavior of killer and sensitive genotypes in several local populations, a prerequisite for studying gamete killer evolution in the wild. This highlights the potential of the Arabidopsis model not only for functional studies of gamete killers but also for investigating their evolutionary trajectories at complementary geographical scales.
A Y chromosome-linked genome editor for efficient population suppression in the malaria vector Anopheles gambiae
34215Tolosana, I., Willis, K., Gribble, M. et al., Nature Communications, 16:206. 2025-01-07 09:21:41.
Genetic control – the deliberate introduction of genetic traits to control a pest or vector population – offers a powerful tool to augment conventional mosquito control tools that have been successful in reducing malaria burden but that are compromised by a range of operational challenges. Self-sustaining genetic control strategies have shown great potential in laboratory settings, but hesitancy due to their invasive and persistent nature may delay their implementation. Here, instead, we describe a self-limiting strategy, designed to have geographically and temporally restricted effect, based on a Y chromosome-linked genome editor (YLE). The YLE comprises a CRISPR-Cas9 construct that is always inherited by males yet generates an autosomal dominant mutation that is transmitted to over 90% of the offspring and results in female-specific sterility. To our knowledge, our system represents a pioneering approach in the engineering of the Y chromosome to generate a genetic control strain for mosquitoes. Mathematical modelling shows that this YLE technology is up to seven times more efficient for population suppression than optimal versions of other self-limiting strategies, such as the widely used Sterile Insect Technique or the Release of Insects carrying a Dominant Lethal gene.
Improving the suppressive power of homing gene drive by co-targeting a distant-site female fertility gene
33386Faber, N.R., Xu, X., Chen, J. et al., Nature Communications, 15. 2024-11-04 17:07:21.
Gene drive technology has the potential to address major biological challenges. Well-studied homing suppression drives have been shown to be highly efficient in Anopheles mosquitoes, but for other organisms, lower rates of drive conversion prevent elimination of the target population. To tackle this issue, we propose a gene drive design that has two targets: a drive homing site where drive conversion takes place, and a distant site where cleavage induces population suppression. We model this design and find that the two-target system allows suppression to occur over a much wider range of drive conversion efficiency. Specifically, the cutting efficiency now determines the suppressive power of the drive, rather than the conversion efficiency as in standard suppression drives. We construct a two-target drive in Drosophila melanogaster and show that both components of the gene drive function successfully. However, cleavage in the embryo from maternal deposition as well as fitness costs in female drive heterozygotes both remain significant challenges for both two-target and standard suppression drives. Overall, our improved gene drive design has the potential to ease problems associated with homing suppression gene drives for many species where drive conversion is less efficient.
Engineering Resilient Gene Drives Towards Sustainable Malaria Control: Predicting, Testing and Overcoming Target Site Resistance
33379Ioanna Morianou, Lee Phillimore, Bhavin S. Khatri,, bioRxiv, 2024-11-04 13:56:24.
CRISPR-based gene drives are selfish genetic elements with the potential to spread through entire insect populations for sustainable vector control. Gene drives designed to disrupt the reproductive capacity of females can suppress laboratory populations of the malaria mosquito. However, any suppressive intervention will inevitably exert an evolutionary pressure for resistance. Here, we present a pipeline for the accelerated discovery, engineering, and testing of both natural and drive-induced variants that could reverse gene drive spread. We applied our method to stress-test a highly effective gene drive that has evaded resistance in all laboratory-contained releases to date, known as Ag(QFS)1. We showed that previously undetected resistant alleles can arise at low frequency, and discovered novel, partially resistant alleles that can perturb drive-invasion dynamics. We then engineered next-generation gene drives that can actively remove resistant alleles by targeting several highly conserved and non-overlapping sites in the female-specific exon of the doublesex gene. Our models predict that such gene drive designs could suppress large, natural populations of the malaria mosquito in the field.
The potential of gene drives in malaria vector species to control malaria in African environments
32702Hancock, P.A., North, A., Leach, A.W. et al., Nature Communications, 15. 2024-10-22 14:44:25.
Gene drives are a promising means of malaria control with the potential to cause sustained reductions in transmission. In real environments, however, their impacts will depend on local ecological and epidemiological factors. We develop a data-driven model to investigate the impacts of gene drives that causes vector population suppression. We simulate gene drive releases in sixteen ~ 12,000 km2 areas of west Africa that span variation in vector ecology and malaria prevalence, and estimate reductions in vector abundance, malaria prevalence and clinical cases. Average reductions in vector abundance ranged from 71.6–98.4% across areas, while impacts on malaria depended strongly on which vector species were targeted. When other new interventions including RTS,S vaccination and pyrethroid-PBO bednets were in place, at least 60% more clinical cases were averted when gene drives were added, demonstrating the benefits of integrated interventions. Our results show that different strategies for gene drive implementation may be required across different African settings.
Identification of novel genes responsible for a pollen killer present in local natural populations of Arabidopsis thaliana
32527Anthony Ricou, Matthieu Simon, et al., bioRxiv, 2024-10-15 11:51:17.
Certain genetic elements are qualified as selfish because they favor their transmission to the progeny during reproduction to the detriment of gametes that do not carry them. These elements are widespread in fungi as well as in plants or in animals, and they are made up of two or even three components, which are specific to each species. Therefore, they must be studied on a case-by-case basis. Moreover, understanding how they appear and propagate in local population remains a major issue in evolutionary biology. Here we have characterized, in the model plant Arabidopsis, the three genes involved in such an element, called a pollen killer. This pollen killer targets the mitochondria to cause the death of pollen grains that do not carry it. We investigated the three genes in several hundred genotypes collected worldwide, giving us a global view of their diversity at the species level. We also found that some French local populations contain both sensitive and killer plants, which constitutes an invaluable resource for studying the evolution of a pollen killer in the wild.
Loss-of-function in testis-specific serine/threonine protein kinase triggers male infertility in an invasive moth
32506Wei, Z., Wang, Y., Zheng, K. et al., Communications Biology, 7. 2024-10-08 09:10:05.
Genetic biocontrol technologies present promising and eco-friendly strategies for the management of pest and insect-transmitted diseases. Although considerable advancements achieve in gene drive applications targeting mosquitoes, endeavors to combat agricultural pests have been somewhat restricted. Here, we identify that the testis-specific serine/threonine kinases (TSSKs) family is uniquely expressed in the testes of Cydia pomonella, a prominent global invasive species. We further generated male moths with disrupted the expression of TSSKs and those with TSSKs disrupted using RNA interference and CRISPR/Cas9 genetic editing techniques, resulting in significant disruptions in spermiogenesis, decreased sperm motility, and hindered development of eggs. Further explorations into the underlying post-transcriptional regulatory mechanisms reveales the involvement of lnc117962 as a competing endogenous RNA (ceRNA) for miR-3960, thereby regulating TSSKs. Notably, orchard trials demonstrates that the release of male strains can effectively suppress population growth. Our findings indicate that targeting TSSKs could serve as a feasible avenue for managing C. pomonella populations, offering significant insights and potential strategies for controlling invasive pests through genetic sterile insect technique (gSIT) technology.
Assessment of drive efficiency and resistance allele formation of a homing gene drive in the mosquito Aedes aegypti
32128Xiaozhen Yang, Xuejiao Xu, et al., bioRxiv, 2024-10-03 09:34:32.
Aedes aegypti, known for transmitting viruses such as dengue, zika, and yellow fever, poses a significant public health threat. Conventional insecticides give rise to a range of issues, including ecological contamination and insect resistance. Hence, there is a pressing demand for environmentally friendly, safer, and more efficacious strategies for mosquito control. With the rapid advancement of the CRISPR/Cas9 system in gene function exploration and pest population control, substantial progress has been achieved in utilizing CRISPR/Cas9-based gene drive systems across various mosquito species. Only a few studies on gene drive technology have been conducted in A. aegypti. In this study, we constructed two complete drives for A. aegypti with different Cas9 promoters, each targeting kmo. Our drive based on Pub-Cas9 had limited activity, but one with exu-Cas9 exhibited super-Mendelian inheritance rates of approximately 60%. We observed low but detectable somatic activity of the drive and no evidence of maternally deposited Cas9. Germline resistance allele formation rates were similar to drive conversion rates, but most wild-type alleles in the germline remained uncut. Injections into the exu-Cas9 drive line had 100% knockout efficiency among surviving offspring at three separate target genes. These results support the development and application of novel genetic pest control technologies aimed at combating A. aegypti.
To CRISPR or Not to CRISPR? Ethical Considerations in Gene-Editing Insects
31614Brendan Parent, Meghan Barrett, American Entomologist, 70:54-57. 2024-09-18 21:33:47.
Genetically modified corn has helped feed the world (Hernandes-Lopes et al. 2023). Genetically modified mosquitoes could help eliminate devastating diseases like malaria (Hammond and Galizi 2017). Plainly, gene editing can serve some important human interests. Still, many people object to it. While some concerns have little scientific validity, there are valid ethical concerns that should be addressed. A concern many people share is the potential impact of genetically edited insects on the environment. Gene drives are “selfish” genetic elements that are transmitted to progeny at unusually high rates and thus spread rapidly through populations. As a result, they are capable of modifying an entire population or species. The most widely discussed use of gene drives is in the prevention of malaria, the leading cause of human illness and death in many parts of the world (CDC 2021), where gene drives could be used to control mosquito populations. At present, however, there are no sure-fire strategies to “recall” a gene drive once it has been released (Hammond and Galizi 2017). Given the many unintended environmental impacts of other technological advancements and our uncertainties about the impacts of using gene drives (Ahmad et al. 2022), it makes sense to have similar concerns about this form of gene editing. Granted, it is possible that the extraordinary benefits to human life of such a gene drive, if successful, outweigh the risks of any unintended environmental consequences. This is particularly likely to be true if convincing measures like “terminator genes” can be employed to control genetically edited insect populations in the wild (Hammond et al. 2021). Given the serious promise and ethical concerns of this technology, the National Academy of Sciences has published guidelines for responsible research that include self-governance and government regulation, evaluating gene drives on a case-by-case basis (NASEM 2016). So, while risks of environmental harm are important, we can now see how they might not be decisive: first, the corresponding benefits might be very weighty; second, the risks might be mitigable.
The ultra-selfish gene
31608Mathias Kirk Bonde, Works in Progress, 2024-09-18 20:59:37.
Almost every cell in our bodies contains 23 pairs of chromosomes, which are packages of the DNA and genes that provide the code for producing living things. Sperm and egg cells, however, each contain only one set of chromosomes. This set of chromosomes has been recombined from their parents’ chromosomes, meaning it contains a random mixture of segments from the parents. When a sperm and egg cell fuse, the resulting cell has a pair of each chromosome once again, resulting in 23 pairs. Because the sections of each chromosome to be passed on were selected randomly, any specific gene in a parent has only a 50 percent chance of making it to the next generation. A gene that helps organisms to have more surviving offspring will gradually become more widespread in the population. But some genes have found ways of overriding this process. For example, what if a gene makes the sperm or egg more likely to inherit the section of DNA where the gene itself is located? In that case, the selection process is no longer random, and the gene can spread across the population even if the gene carries no advantage to the animal’s fitness.
Deployment of tethered gene drive for confined suppression in continuous space requires avoiding drive wave interference
31622Ruobing Feng, Jackson Champer, Molecular Ecology, 33. 2024-09-16 21:13:35.
Gene drives have great potential for suppression of pest populations and removal of exotic invasive species. CRISPR homing suppression drive is a powerful but unconfined drive, posing risks of uncontrolled spread. Thus, developing methods for confining a gene drive is of great significance. Tethered drive combines a confined system such as Toxin-Antidote Recessive Embryo drive with a strong drive such as a homing suppression drive. It can prevent the homing drive from spreading beyond the confined drive and can be constructed readily, giving it good prospects for future development. However, we have found that care must be taken when deploying tethered drive systems in some scenarios. Simulations of tethered drive in a panmictic population model reveal that successful deployment requires a proper release ratio between the two components, tailored to prevent the suppression drive from eliminating the confined system before it has the chance to spread. Spatial models where the population moves over a one-dimensional landscape display a more serious phenomenon of drive wave interference between the two tethered drive components. If the faster suppression drive wave catches up to the confined drive wave, success is still possible, but it is dependent on drive performance and ecological parameters. Two-dimensional simulations further restrict the parameter range for drive success. Thus, careful consideration must be given to drive performance and ecological conditions, as well as specific release proposals for potential application of tethered drive systems.
Population suppression by release of insects carrying a dominant sterile homing gene drive targeting doublesex in Drosophila
34760Chen, W., Guo, J., Liu, Y. et al., Nature Communications, 15. 2024-09-14 09:17:45.
CRISPR homing gene drives can suppress pest populations by targeting female fertility genes, converting wild-type alleles into drive alleles in the germline of drive heterozygotes. fsRIDL (female-specific Release of Insects carrying a Dominant Lethal) is a self-limiting population suppression strategy involving continual release of transgenic males carrying female lethal alleles. Here, we propose an improved pest suppression system called “Release of Insects carrying a Dominant-sterile Drive” (RIDD), combining performance characteristics of homing drive and fsRIDL. We construct a split RIDD system in Drosophila melanogaster by creating a 3-gRNA drive disrupting the doublesex female exon. Drive alleles bias their inheritance in males, while drive alleles and resistance alleles formed by end-joining cause dominant female sterility. Weekly releases of RIDD males progressively suppressed and eventually eliminated cage populations. Modeling shows that RIDD is substantially stronger than SIT and fsRIDL. RIDD is also self-limiting, potentially allowing targeted population suppression.
New genetic-editing technique to alter the traits and fates of wild populations
31454Lori Dajose, CalTech, 2024-09-03 21:38:08.
Gene drives are a common technology used to insert a novel gene into a population—for example, to make mosquitos resistant to malaria. They can also be used to modify existing genes, such as making herbicide-resistant weeds susceptible to herbicides once again or even to suppress invasive populations. However, gene drives often face social concerns and regulatory challenges because they involve the spread of transgenes (genes that have been transferred and integrated into an organism's DNA) to high frequency. The new technique, called an Allele Sail, uses the CRISPR/Cas9 genome-editing technology to introduce a targeted "editor" into a population at low frequency. This editor itself does not increase in frequency as organisms reproduce, however, any organism that mates with an editor-carrying organism will become altered at the genomic position targeted by the editor, passing the altered version (called an allele) of the gene down to its own offspring. In this way, the technique mimics the natural genetic process of passing down genes and mutations, and can be used in a wider range of species than traditional gene drive approaches. "Imagine you have a big room of bouncing balls, most of them white but a few are red," says Bruce Hay, professor of biology and biological engineering. "Any time a red ball—the editor—bumps into a white ball, it turns the white ball pink—the edit. As the balls bounce around, over time, more and more of them turn pink."
Advancements and Future Prospects of CRISPR-Cas-Based Population Replacement Strategies in Insect Pest Management
31330Zhao Y, Li L, Wei L, Wang Y, Han Z., Insects, 15. 2024-09-03 18:39:10.
Many insects are categorized as agricultural pests due to their ability to transmit diseases and damage crops, which results in significant economic losses. Scientists have proposed two main pest control strategies: population suppression, aimed at reducing the size or distribution of pest populations, and population replacement, which involves introducing genetically modified populations to replace wild pests after an initial release. Typically, population replacement strategies use gene drive systems to spread beneficial traits throughout the target population. Current promising gene drive systems include homing endonuclease genes (HEGs), Wolbachia, maternal-effect dominant embryonic arrest (Medea), and newly adapted CRISPR/Cas genome editing systems. This review provides an overview of the recent advancements in population replacement, including insights into the development, testing, and safe implementation of CRISPR-Cas-based gene drive techniques from laboratory settings to field applications. It also discusses recent developments, identifies research gaps, and offers a comprehensive analysis of genetic control strategies for insect pests.
World Mosquito Day: Gene Drives and CRISPR Technology
31205Public Health On Call, YouTube, 2024-08-27 14:16:24.
About this episode: World Mosquito Day, observed annually on August 20th, commemorates British doctor Sir Ronald Ross's discovery in 1897 that female Anopheles mosquitoes transmit malaria to humans. More than a century later, major advancements like genetically modifying mosquitoes—AKA gene drives—have the potential to reduce malaria cases and deaths dramatically, but not without hurdles. This special episode is an extended version of Malaria Minute, a podcast from the Johns Hopkins Malaria Research Institute.
Generating and testing the efficacy of reagents for CRISPR/Cas9 homology directed repair-based manipulations in Tribolium
31183Hannah C Markley, Kennedy J Helms, Megan Maar, Gabriel E Zentner, Michael J Wade, Andrew C Zelhof, Journal of Insect Science, 24. 2024-08-25 20:44:44.
CRISPR/Cas9 manipulations are possible in many insects and ever expanding. Nonetheless, success in one species and techniques developed for it are not necessarily applicable to other species. As such, the development and expansion of CRISPR-based (clustered regularly interspaced short palindromic repeats) genome-editing tools and methodologies are dependent upon direct experimentation. One useful technique is Cas9-dependent homologous recombination, which is a critical tool for studying gene function but also for developing pest related applications like gene drive. Here, we report our attempts to induce Cas9 homology directed repair (HDR) and subsequent gene drive in Tribolium castaneum (Herbst; Insecta: Coleoptera: Tenebrionidae). Utilizing constructs containing 1 or 2 target gRNAs in combination with Cas9 under 2 different promoters and corresponding homology arms, we found a high incidence of CRISPR/Cas9 induced mutations but no evidence of homologous recombination. Even though the generated constructs provide new resources for CRISPR/Cas9 modification of the Tribolium genome, our results suggest that additional modifications and increased sample sizes will be necessary to increase the potential and detection for HDR of the Tribolium genome.
Gene drives: an alternative approach to malaria control?
30958Naidoo, K., Oliver, S.V., Gene Therapy, 2024-07-25 20:08:16.
Genetic modification for the control of mosquitoes is frequently touted as a solution for a variety of vector-borne diseases. There has been some success using non-insecticidal methods like sterile or incompatible insect techniques to control arbovirus diseases. However, control by genetic modifications to reduce mosquito populations or create mosquitoes that are refractory to infection with pathogens are less developed. The advent of CRISPR-Cas9-mediated gene drives may advance this mechanism of control. In this review, use and progress of gene drives for vector control, particularly for malaria, is discussed. A brief history of population suppression and replacement gene drives in mosquitoes, rapid advancement of the field over the last decade and how genetic modification fits into the current scope of vector control are described. Mechanisms of alternative vector control by genetic modification to modulate mosquitoes’ immune responses and anti-parasite effector molecules as part of a combinational strategy to combat malaria are considered. Finally, the limitations and ethics of using gene drives for mosquito control are discussed.
Assessing CRISPR/Cas9 potential in SDG3 attainment: malaria elimination—regulatory and community engagement landscape
30888Snuzik, A., Malaria Journal, 23. 2024-07-07 21:42:05.
Elimination of malaria has become a United Nations member states target: Target 3.3 of the sustainable development goal no. 3 (SDG3). Despite the measures taken, the attainment of this goal is jeopardized by an alarming trend of increasing malaria case incidence. Globally, there were an estimated 241 million malaria cases in 2020 in 85 malaria-endemic countries, increasing from 227 million in 2019. Malaria case incidence was 59, which means effectively no changes in the numbers occurred, compared with the baseline 2015. Jennifer Doudna—co-inventor of CRISPR/Cas9 technology—claims that CRISPR holds the potential to lessen or even eradicate problems lying in the centre of SDGs. On the same note, CRISPR/Cas9-mediated mosquito-targeting gene drives (MGD) are perceived as a potential means to turn this trend back and put momentum into the malaria elimination effort. This paper assessed two of the critical elements of the World Health Organization Genetically modified mosquitoes (WHO GMM) Critical Pathway framework: the community and stakeholders’ engagement (inability to employ widely used frameworks, segmentation of the public, ‘bystander’ status, and guidelines operationalization) and the regulatory landscape (lex generali, ‘goldilocks dilemma’, and mode of regulation) concerning mosquito-oriented gene drives (MGD) advances. Based on the assessment findings, the author believes that CRISPR/Cas-9-mediated MGD will not contribute to the attainment of SDG3 (Target 3.3), despite the undisputable technology’s potential. This research pertains to the state of knowledge, legal frameworks, and legislature, as of November 2022.
99% gene transmission possible, China’s CRISPR tool boosts food security
30872Gairika Mitra, Interesting Engineering, 2024-07-02 12:36:50.
Chinese scientists have engineered a solution by which they could bypass natural plant gene inheritance. They aim to deploy a CRISPR-based gene editing system to help the transmission of preferred genes even when they aren’t suitable for a plant. The scientists devised a system that would use both a toxin and an antidote which would directly affect the male plant germline. Through this process, the researchers could overcome the natural Mendelian transmission rate. This can help increase the gene transmission rates up to 99% over two generations.
Chinese scientists find natural selection loophole that could help transform food security
30867Victoria Bela, South China Morning Post, 2024-07-02 12:29:51.
Scientists in China have found a way to bypass natural plant gene inheritance, by using a CRISPR-based gene editing system to boost the transmission of preferred genes even when they are detrimental to a plant. By harnessing a system that uses both a toxin and an antidote to target the male plant germline, the scientists were able to overcome the natural Mendelian transmission rate, achieving gene transmission rates of up to 99 per cent over two generations. “Facing diverse challenges such as threats to food security from agricultural weeds and the environmental crisis of invasive plants, the genetic manipulation of wild plant populations has emerged as a potentially powerful and transformative strategy,” the team wrote in a paper published in the peer-reviewed journal Nature Plants on Monday.
A New CRISPR-Driven Technology for Gene Drive in Plants
30849Lori Dajose, CalTech, 2024-06-28 11:49:31.
Spreading a specific genetic trait through a population, even if that trait does not benefit those who carry it, is the purpose of a "gene drive." Gene drives can be used for many different applications. These are divided into two broad categories: population modification and population suppression. Population modification can make mosquitos immune to, and therefore unable to spread, malaria, or make a crop more heat-tolerant in anticipation of climate change. Population suppression can be used to bring about local reduction or elimination of a weed or invasive species. But any gene editing program needs to have strict built-in controls to keep the modifications localized to a specific area and to prevent other species from accidentally inheriting modified genes. Now, Caltech researchers have developed a new gene drive technology, called ClvR (pronounced "cleaver"), that can be specifically customized to plant species, preventing accidental gene editing in cross-pollination situations. Crucially, the technology can be designed to be self-limiting, only spreading the desired genes for a limited number of generations, thereby limiting their spread in time and space. The work is the first engineered gene drive in plants and the first to enable species-specific modification as well as the first to act at the level of plant sex cells.
Germline Cas9 promoters with improved performance for homing gene drive
30488Du, J., Chen, W., Jia, X. et al., Nature Communications, 15:4560. 2024-06-04 16:50:42.
Gene drive systems could be a viable strategy to prevent pathogen transmission or suppress vector populations by propagating drive alleles with super-Mendelian inheritance. CRISPR-based homing gene drives convert wild type alleles into drive alleles in heterozygotes with Cas9 and gRNA. It is thus desirable to identify Cas9 promoters that yield high drive conversion rates, minimize the formation rate of resistance alleles in both the germline and the early embryo, and limit somatic Cas9 expression. In Drosophila, the nanos promoter avoids leaky somatic expression, but at the cost of high embryo resistance from maternally deposited Cas9. To improve drive efficiency, we test eleven Drosophila melanogaster germline promoters. Some achieve higher drive conversion efficiency with minimal embryo resistance, but none completely avoid somatic expression. However, such somatic expression often does not carry detectable fitness costs for a rescue homing drive targeting a haplolethal gene, suggesting somatic drive conversion. Supporting a 4-gRNA suppression drive, one promoter leads to a low drive equilibrium frequency due to fitness costs from somatic expression, but the other outperforms nanos, resulting in successful suppression of the cage population. Overall, these Cas9 promoters hold advantages for homing drives in Drosophila species and may possess valuable homologs in other organisms.
Altering traits and fates of wild populations with Mendelian DNA sequence modifying Allele Sails
29102Michelle L. Johnson, Bruce A. Hay, Maciej Maselko, Nature Communications, 15. 2024-04-16 09:23:41.
Population-scale genome editing can be used to alter the composition or fate of wild populations. One approach to achieving these aims utilizes a synthetic gene drive element—a multi-gene cassette—to bring about an increase in the frequency of an existing allele. However, the use of gene drives is complicated by the multiple scientific, regulatory, and social issues associated with transgene persistence and gene flow. Alternatives in which transgenes are not driven could potentially avoid some of these issues. Here we propose an approach to population scale gene editing using a system we refer to as an Allele Sail. An Allele Sail consists of a genome editor (the Wind) that introduces DNA sequence edits (the Sail) at one or more sites, resulting in progeny that are viable and fertile. The editor, such as a sequence-specific nuclease, or a prime- or base-editor, is inherited in a Mendelian fashion. Meanwhile, the edits it creates experience an arithmetic, Super-Mendelian increase in frequency. We explore this system using agent-based modeling, and identify contexts in which a single, low frequency release of an editor brings edits to a very high frequency. We also identify conditions in which manipulation of sex determination can be used to bring about population suppression. Current regulatory frameworks often distinguish between transgenics as genetically modified organisms (GMOs), and their edited non-transgenic progeny as non-GMO. In this context an Allele Sail provides a path to alter traits and fates of wild populations in ways that may be considered more acceptable.
Alpha-mannosidase-2 modulates arbovirus infection in a pathogen- and Wolbachia-specific manner in Aedes aegypti mosquitoes
29035Nadya Urakova, Renuka E. Joseph, Allyn Huntsinger, Vanessa M. Macias, Matthew J. Jones, Leah T. Sigle, Ming Li, Omar S. Akbari, Zhiyong Xi, Konstantinos Lymperopoulos, Richard T. Sayre, Elizabeth A. McGraw, Jason L. Rasgon, Insect Molecular Biology, 2024-03-19 15:50:02.
Multiple Wolbachia strains can block pathogen infection, replication and/or transmission in Aedes aegypti mosquitoes under both laboratory and field conditions.
CRISPR could eradicate horrific parasite that’s killing cattle
28993Kristin Houser, Freethink, 2024-03-11 10:19:23.
Uruguay is developing a CRISPR gene drive to eradicate the New World screwworm, a parasitic fly that kills cattle in a painful, grisly fashion. Releasing it into the wild would have risks, but if it works, it could help rid South America of this horrific agricultural pest. The screwworm fly lays its eggs on living creatures — often livestock — and once they hatch, the larvae eat into the animal’s flesh for about a week, before emerging from the tunnel they created and flying away. “We know that it’s horrendously painful, because people get affected by this, and the standard of treatment is you give them morphine immediately so that surgeons can cut the things out — because it’s just that painful; it’s unbelievably agonizing,” Kevin Esvelt, a biologist at the MIT Media Lab, told the 80,000 Hours podcast in 2023. Aside from being painful, screwworm infestations of livestock are incredibly costly. In the 1950s, the US meat and dairy industries were losing an estimated $200 million per year to the pests — that’d be about $2.3 billion today. Screwworms are no longer a problem for American farmers thanks to the USDA. In 1955, it set out to eradicate the screwworm in the US by irradiating the insects’ larvae, which made adults sterile. Infertile males could then be released into infested areas to mate with females, which wouldn’t produce any offspring.
Un1Cas12f1 and Cas9 gene drive in HSV1: viruses that ‘infect’ viruses
28972Qiaorui Yao, Zhuangjie Lin, Keyuan Lai, Xianying Zeng, Guanxiong Lei, Tongwen Zhang, Hongsheng Dai, bioRxiv, 2024-03-05 14:11:19.
Synthetic CRISPR-Cas9 gene drive has been developed as a potential tool to control harmful species. However, Cas9 gene drive faces high resistance rate and mitigation strategies developed so far are difficult to implement. Furthermore, studying the resistance to gene drive is time consuming and challenging in higher organisms. We here tackled these two challenges simultaneously by generating Cas9 and Un1Cas12f1 gene drive in a fast-replicating DNA virus, HSV1. We assessed the transmission dynamics and resistance formation through phenotypical staining and next-generation sequencing, and demonstrated that HSV1 supported fast and effective transmission of gene drives, and the Un1Cas12f1 gene drives yielded greater conversion and lower resistance than did the Cas9 gene drives. This positions the Un1Cas12f1 gene drive as a promising alternative, and HSV1 emerges as a dependable and swift platform for gene drive assessment. The gene drive viruses function like pathogens that specifically infect viruses, offering potential applications in attenuating viral infections.
CRISPR-mediated germline mutagenesis for genetic sterilization of Anopheles gambiae males
28970Smidler, A.L., Marrogi, E., Kauffman, J. et al., Scientific Reports, 14. 2024-03-05 13:54:40.
Rapid spread of insecticide resistance among anopheline mosquitoes threatens malaria elimination efforts, necessitating development of alternative vector control technologies. Sterile insect technique (SIT) has been successfully implemented in multiple insect pests to suppress field populations by the release of large numbers of sterile males, yet it has proven difficult to adapt to Anopheles vectors. Here we outline adaptation of a CRISPR-based genetic sterilization system to selectively ablate male sperm cells in the malaria mosquito Anopheles gambiae. We achieve robust mosaic biallelic mutagenesis of zero population growth (zpg, a gene essential for differentiation of germ cells) in F1 individuals after intercrossing a germline-expressing Cas9 transgenic line to a line expressing zpg-targeting gRNAs. Approximately 95% of mutagenized males display complete genetic sterilization, and cause similarly high levels of infertility in their female mates. Using a fluorescence reporter that allows detection of the germline leads to a 100% accurate selection of spermless males, improving the system. These males cause a striking reduction in mosquito population size when released at field-like frequencies in competition cages against wild type males. These findings demonstrate that such a genetic system could be adopted for SIT against important malaria vectors.
Gene Drive Systems To Control Aedes Aegypti Mosquitoes Make Headway
28966Joshua Ang, Outreach Network for Gene Drive Research, 2024-03-05 13:15:42.
Aedes aegypti mosquitoes are known vectors of several diseases, including dengue, chikungunya, yellow fever, and Zika, which impact millions of people worldwide each year. The effectiveness of existing insecticide-based methods to control this mosquito is threatened by growing insecticide resistance, underscoring the need to develop new approaches. The advent of CRISPR/Cas9 genome editing has reshaped the research and development landscape of new potential vector control tools, leading researchers to explore novel approaches, such as gene drive technologies. In the past few years, gene drive technologies have gained remarkable traction, particularly for their success in controlling major malaria mosquito vectors in laboratory settings. A gene drive is able to bias its own inheritance, facilitating the spread of a specific trait through a target population. This super-Mendelian pattern of propagation makes gene drive technology an efficient and cost-effective potential new method to control mosquitoes that transmit disease.
Revolutionizing Livestock Biosecurity: Using CRISPR Technology to Combat the New World Screwworm
28963Dr. Jessica Nelson, Medriva, 2024-03-05 13:07:55.
The New World screwworm, a persistent parasite responsible for significant damage to the global livestock industry, may soon meet its match. Researchers at Uruguay's National Institute of Agricultural Research (INIA) have developed a gene drive using CRISPR technology to combat this destructive pest. By manipulating the reproductive process of the screwworm fly, INIA scientists aim to cause a population crash, thereby reducing the parasite's devastating impact on the livestock industry. CRISPR gene drive technology offers a potentially more efficient and powerful solution compared to previous methods, such as the sterile insect technique (SIT) used by the US. Unlike traditional techniques, CRISPR gene drives aim to spread fertility-damaging genes throughout the screwworm population, causing a significant decrease in their numbers. The process works by making female screwworms sterile. The ultimate goal is to release gene-edited male screwworm flies into the wild. These males will mate with females, passing on the gene drive and leading to a population crash of the screwworm fly. This innovative approach has shown promise in caged trials and is currently being tested further in the INIA labs.
Uruguay wants to use gene drives to eradicate devastating screwworms
28961Abdullahi Tsanni, MIT Technology Review, 2024-03-05 12:53:17.
On a warm, sunny day in Montevideo, Uruguay, the air is smogless and crisp. Inside a highly secured facility at the National Institute of Agricultural Research (INIA) are a sophisticated gene gun, giant microscopes, and tens of thousands of gene-edited flies, their bright blue wings fluttering against the walls of their small, white, netted cages. These flies—shown to me on video by an INIA veterinarian, Alejo Menchaca—are a new weapon that may soon be unleashed against an enemy that kills cattle and costs the livestock industry millions of dollars every year: the New World screwworm, a parasite common in parts of South America and the Caribbean. When a female screwworm fly attacks cattle, it lays eggs, which hatch and turn into worm-like larvae that screw down into the host animal, feeding on flesh along their way and damaging the animal’s skin. Left untreated, the animals eventually die in excruciating agony. But Menchaca and colleagues have a plan. Using the genome-editing system CRISPR, they’ve developed what’s known as a gene drive, a type of genetic element that manipulates the reproductive process to spread farther and faster than an ordinary gene. They are about to move into the next stage of caged trials in the lab, with a view to eventually using the genetic tool to decimate the screwworm fly population. In collaboration with Institut Pasteur de Montevideo, they have received a $450,000 grant from the Inter-American Development Bank (IDB) for the research.
Genome editing in pests: basic science to applications
28908Chen, X., Palli, S.R., Journal of Pest Science, 2024-02-20 15:39:21.
Recent developments in sequencing technologies produced enormous data on gene sequences and the identity of genes in many pest insects and disease vectors. However, the function of many of these genes is unknown. Functional genomics studies to uncover gene function in pest insects are urgently needed. RNA interference methods could be used in some insects but not most due to their variable efficiency among insect pests. Recently developed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system of genome editing method is being developed for use in many insect pests. This technology has already been demonstrated to function in more than 40 insect pest species from seven orders and has contributed to advances in pest biology and the development of improved pest management methods. This review summarizes recent results of CRISPR/Cas9 technology developments and their contributions to advancing the basic and applied science of insect pests and disease vectors.
Transforming malaria prevention and control: the prospects and challenges of gene drive technology for mosquito management
28861Yusuf Amuda Tajudeen, Habeebullah Jayeola Oladipo, Iyiola Olatunji Oladunjoye, Muhammad Kamaldeen Oladipo, Hameedat Damilola Shittu, Imam-Fulani Abdulmumeen, Abdullateef Opeyemi Afolabi and Mona Said El-Sherbini, Annals of Medicine, 55. 2024-01-30 17:32:22.
In the era of insecticides and anti-malarial drug resistance, gene drive technology holds considerable promise for malaria control. Gene drive technology deploys genetic modifications into mosquito populations to impede their ability to transmit the malaria parasite. This can be either through the disruption of an essential mosquito gene or the association of gene drive with a desirable effector gene. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a gene editing tool that precisely modifies mosquito vector DNA sequences and curtails the rate of pathogen transmission. A comprehensive search was conducted in the SCOPUS and MEDLINE databases (via PubMed) until October 2023. The keywords used were related to the principles and mechanisms of gene drive technology, its advantages, and disadvantages, and its ethical and regulatory considerations in sustainable malaria eradication. The development of gene drive enables the preferential inheritance of specific genes in targeted mosquitoes, potentially obstructing the transmission of the Plasmodium parasite. This technology was also studied for the control of other vector-borne diseases such as dengue and chikungunya viruses. Despite its experimental superiority over other traditional methods such as insecticide-treated nets and insecticide sprays, the long-term dynamic interplay of mutation and resistance poses challenges for gene drive efficiency in sustainable malaria control. This commentary elucidates the underlying mechanisms and principles of gene drive technology, underscoring its promise and challenges as a novel strategy to curtail malaria prevalence. Although the release of such genetically modified mosquitoes into the natural environment would result in the eradication of the locally targeted species of mosquitoes, the complete eradication of the entire species remains questionable. Thus, the practical application raises significant ethical and regulatory concerns for further research and risk assessment, including the risk of gene drive spreading to nontarget species in the wider theatre of biodiverse species.
A multiplexed, confinable CRISPR/Cas9 gene drive can propagate in caged Aedes aegypti populations
28857Anderson, M.A.E., Gonzalez, E., Edgington, M.P. et al., Nature Communications, 15. 2024-01-30 16:38:51.
Aedes aegypti is the main vector of several major pathogens including dengue, Zika and chikungunya viruses. Classical mosquito control strategies utilizing insecticides are threatened by rising resistance. This has stimulated interest in new genetic systems such as gene drivesHere, we test the regulatory sequences from the Ae. aegypti benign gonial cell neoplasm (bgcn) homolog to express Cas9 and a separate multiplexing sgRNA-expressing cassette inserted into the Ae. aegypti kynurenine 3-monooxygenase (kmo) gene. When combined, these two elements provide highly effective germline cutting at the kmo locus and act as a gene drive. Our target genetic element drives through a cage trial population such that carrier frequency of the element increases from 50% to up to 89% of the population despite significant fitness costs to kmo insertions. Deep sequencing suggests that the multiplexing design could mitigate resistance allele formation in our gene drive system.
Gene drive and genetic sex conversion in the global agricultural pest Ceratitis capitata
28802Meccariello, A., Hou, S., Davydova, S. et al., Nature Communications, 15:372. 2024-01-15 17:13:20.
Homing-based gene drives are recently proposed interventions promising the area-wide, species-specific genetic control of harmful insect populations. Here we characterise a first set of gene drives in a tephritid agricultural pest species, the Mediterranean fruit fly Ceratitis capitata (medfly). Our results show that the medfly is highly amenable to homing-based gene drive strategies. By targeting the medfly transformer gene, we also demonstrate how CRISPR-Cas9 gene drive can be coupled to sex conversion, whereby genetic females are transformed into fertile and harmless XX males. Given this unique malleability of sex determination, we modelled gene drive interventions that couple sex conversion and female sterility and found that such approaches could be effective and tolerant of resistant allele selection in the target population. Our results open the door for developing gene drive strains for the population suppression of the medfly and related tephritid pests by co-targeting female reproduction and shifting the reproductive sex ratio towards males. They demonstrate the untapped potential for gene drives to tackle agricultural pests in an environmentally friendly and economical way.
Gene Drives: Mechanisms and Key Research, Explained
28795Julia Bauman, 60 Second Science, 2024-01-15 16:59:40.
A technical primer on CRISPR-based gene drives, which hold massive potential for mitigating the harms invoked by some species. We cover what a gene drive is, how it works at the genetic level, and summarize key safety & efficiency features developed in recent years.
CRISPR engineered viruses could render other viruses harmless
28784Michael Le Page, New Scientist, 2023-12-29 13:14:45.
A virus genetically engineered to spread its DNA to other viruses via CRISPR gene editing has done exactly that in tests in mice. The hope is that these viruses could alter others, such as herpes, in a way that prevents them from causing symptoms. “It’s a new technology,” says team member Marius Walter at the Fred Hutch Cancer Center in Seattle, Washington. “Can we bring it to people? That’s a long way ahead, we have a lot of work to do, but I think this is an exciting technology." This approach is known as a gene drive, when a bit of selfish DNA somehow manages to get passed down to a higher proportion of offspring than normal. This means gene drives can spread through a population even if they are disadvantageous.
Gene drives, mosquitoes, and ecosystems: An interdisciplinary approach to emerging ethical concerns
28778Ricardo D. Moreno, Luca Valera, Cristián Borgoño, Juan Carlos Castilla, José Luis Riveros, Frontiers in Environmental Science, 11. 2023-12-28 22:14:12.
Gene drives are genetic elements that in sexually reproducing organisms spread faster than those transmitted through a Mendelian fashion. Since gene drives can be engineered to modify different aspects of physiology and reproduction, they have been proposed as a new and revolutionary tool to control vector-borne diseases, particularly those transmitted by the genera Anopheles and Aedes (Culicidae), such as malaria, Dengue and Zika virus. This approach may impact on human health by lowering the transmission of such devastating diseases. However, the release of genetically modified mosquitos (or other species) into the environment raises a series of questions related to the still incipient technology and our present understanding of the complex structure and dynamics of terrestrial and aquatic ecosystems. Moreover, there are ethical concerns about human interventions in natural ecosystems that may eventually impact our way of living or the ecosystems themselves. This work is an interdisciplinary approach that analyzes from a biological, philosophical, and theological perspective the potential ecological impacts on natural environments of the release of genetically modified species, focusing on gene drive-modified mosquitos. It includes theological approach from a Catholic point of view (although it could be easily shared by other Christians) because we hold that world religions give valuable insights even though not everyone may share their groundings. We conclude that the focal problem is the relationship between humans and nature, and the release of genetically modified species may change this relationship unpredictably. However, given the complex interactions in ecosystems, new approaches such as Earth Stewardship principles could provide new and more widely accepted answers involving biological, philosophical, and theological concepts that will help engaging all relevant actors to make a better world.
Advancements in Gene Editing: Using CRISPR-Cas9 and Gene Drive Technology to Neutralize Viruses
28765Anonymous, News Directory 3, 2023-12-27 20:03:12.
Researchers from the US Fred Hutchinson Cancer Research Center recently published research results in the international academic journal “Nature” demonstrating that herpes virus type 1 (HSV-1) was neutralized using “Gene Drive” technology . Gene drive refers to a technology that changes the genes of a specific organism and affects the entire organism. The international academic journal Science said: “Scientists highly appreciate the potential of gene drive technology” and added: “It could be an opportunity to pave the way for modifying viral populations through genetic manipulation and using it as a new treatment."
CRISPR/Cas9: a cutting-edge solution for combatting the fall armyworm, Spodoptera frugiperda
28724Gouda, M.N.R., Jeevan, H., Shashank, H.G., Molecular Biology Reports, 51. 2023-12-19 12:15:58.
The utilization of CRISPR/Cas9 in Spodoptera frugiperda, commonly known as fall armyworm, presents a groundbreaking avenue for pest management. With its ability to precisely modify the insect’s genome, CRISPR/Cas9 offers innovative strategies to combat this destructive pest. The application of CRISPR/Cas9 in S. frugiperda holds immense potential. It enables the identification and functional analysis of key genes associated with its behavior, development, and insecticide resistance. This knowledge can unveil novel target sites for more effective and specific insecticides. Additionally, CRISPR/Cas9 can facilitate the development of population control methods by disrupting vital genes essential for survival. However, challenges such as off-target effects and the efficient delivery of CRISPR/Cas9 components remain. Addressing these obstacles is vital to ensure accurate and reliable results. Furthermore, ethical considerations, biosafety protocols, and regulatory frameworks must be integral to the adoption of this technology. Looking forward, CRISPR/Cas9-based gene drive systems hold the potential to promulgate desirable genetic traits within S. frugiperda populations, offering a sustainable and eco-friendly approach. This could curtail their reproductive capabilities or make them more susceptible to certain interventions. In conclusion, CRISPR/Cas9 presents a transformative platform for precise and targeted pest management in S. frugiperda. By deciphering the insect’s genetic makeup and developing innovative strategies, we can mitigate the devastating impact of fall armyworm on agriculture while ensuring environmental sustainability.
Upper Bound on the Mutational Burden Imposed by a CRISPR-Cas9 Gene-Drive Element
28722Michael S. Overton, Sean E. Guy, Xingsen Chen, Alena Martsul, Krypton Carolino, Omar S. Akbari, Justin R. Meyer, Sergey Kryazhimskiy, bioRxiv, 2023-12-19 12:04:20.
CRISPR-Cas9 gene drives (CCGDs) are powerful tools for genetic control of wild populations, useful for eradication of disease vectors, conservation of endangered species and other applications. However, Cas9 alone and in a complex with gRNA can cause double-stranded DNA breaks at off-target sites, which could increase the mutational load and lead to loss of heterozygosity (LOH). These undesired effects raise potential concerns about the long-term evolutionary safety of CCGDs, but the magnitude of these effects is unknown. To estimate how the presence of a CCGD or a Cas9 alone in the genome affects the rates of LOH events and de novo mutations, we carried out a mutation accumulation experiment in yeast Saccharomyces cerevisiae. Despite its substantial statistical power, our experiment revealed no detectable effect of CCGD or Cas9 alone on the genome-wide rates of mutations or LOH events, suggesting that these rates are affected by less than 30%. Nevertheless, we found that Cas9 caused a slight but significant shift towards more interstitial and fewer terminal LOH events, and the CCGD caused a significant difference in the distribution of LOH events on Chromosome V. Taken together, our results show that these genetic elements impose a weak and likely localized additional mutational burden in the yeast model. Although the mutagenic effects of CCGDs need to be further evaluated in other systems, our results suggest that the effect of CCGDs on off-target mutation rates and genetic diversity may be acceptable.
Gene Drive: Engineered viruses take on their own kind in a new study
28711Rizwan Choudhury, Interesting Engineering, 2023-12-18 09:27:39.
Scientists are exploring a radical idea to combat viral infections: use viruses against themselves. They are testing whether introducing modified viruses into people with the same natural viruses can spread a gene that destroys the infection. This has yet to be done successfully in animals, but a study has shown it is possible in theory. The technique uses gene drives, tools that use CRISPR, a gene editing system, to speed up gene transmission in the offspring. Gene drives have been used to alter animals like mice and mosquitoes to reduce their numbers. But a recent study showed that gene drives can also work with herpesvirus-1 (HSV-1), which causes cold sores. When mice were infected with both normal and modified herpesviruses, the gene drive changed up to 90% of the viruses, which could prevent the infection from causing symptoms. Another study achieved similar results with HSV-1 in cells in the lab.
Transformative Approaches for Sustainable Weed Management: The Power of Gene Drive and CRISPR-Cas9
28677Kumam, Y.; Trick, H.N.; Vara Prasad, P.V.; Jugulam, M., Genes, 14. 2023-12-06 13:31:23.
Weeds can negatively impact crop yields and the ecosystem’s health. While many weed management strategies have been developed and deployed, there is a greater need for the development of sustainable methods for employing integrated weed management. Gene drive systems can be used as one of the approaches to suppress the aggressive growth and reproductive behavior of weeds, although their efficacy is yet to be tested. Their popularity in insect pest management has increased, however, with the advent of CRISPR-Cas9 technology, which provides specificity and precision in editing the target gene. This review focuses on the different types of gene drive systems, including the use of CRISPR-Cas9-based systems and their success stories in pest management, while also exploring their possible applications in weed species. Factors that govern the success of a gene drive system in weeds, including the mode of reproduction, the availability of weed genome databases, and well-established transformation protocols are also discussed. Importantly, the risks associated with the release of weed populations with gene drive-bearing alleles into wild populations are also examined, along with the importance of addressing ecological consequences and ethical concerns.
Viruses that ‘infect’ viruses: Cas12f1 and Cas9 gene drive in HSV1
28673Hongsheng Dai, Qiaorui Yao, Zhuangjie Lin, Keyuan Lai, Xianyin Zeng, Guangxiong Lei, Tongwen Zhang, bioRxiv, 2023-12-06 12:08:32.
Cas9-based synthetic gene drives constitute some minimal elements capable of editing DNA with sequence specificity. However, they face high resistance rate and mitigation strategies developed so far are difficult to implement. Here, we engineered herpes simplex virus type 1 (HSV1) as a vector for gene drive, generated viruses carrying either Cas9 or Cas12f1 gene drives, and compared their performance in spreading the gene drive and causing resistance. Based on phenotypical staining and NGS sequencing, we showed that HSV1 supported fast and effective transmission of gene drive, and Cas12f1 gene drive yielded higher conversion and lower resistance than Cas9 gene drive. Cas12f1 gene drive is a promising alternative to Cas9 gene drive, and HSV1 is a reliable and fast platform to evaluate gene drive. Gene drive viruses worked like pathogens that specifically infect viruses and could potentially be used to attenuate viral infection.
A population modification gene drive targeting both Saglin and Lipophorin impairs Plasmodium transmission in Anopheles mosquitoes
28671Emily I Green, Etienne Jaouen, Dennis Klug, Roenick Proveti Olmo, Amandine Gautier, Stéphanie Blandin, Eric Marois, eLife, 12. 2023-12-06 11:55:36.
Lipophorin is an essential, highly expressed lipid transport protein that is secreted and circulates in insect hemolymph. We hijacked the Anopheles coluzzii Lipophorin gene to make it co-express a single-chain version of antibody 2A10, which binds sporozoites of the malaria parasite Plasmodium falciparum. The resulting transgenic mosquitoes show a markedly decreased ability to transmit Plasmodium berghei expressing the P. falciparum circumsporozoite protein to mice. To force the spread of this anti-malarial transgene in a mosquito population, we designed and tested several CRISPR/Cas9-based gene drives. One of these is installed in, and disrupts, the pro-parasitic gene Saglin and also cleaves wild-type Lipophorin, causing the anti-malarial modified Lipophorin version to replace the wild type and hitch-hike together with the Saglin drive. Although generating drive-resistant alleles and showing instability in its gRNA-encoding multiplex array, the Saglin-based gene drive reached high levels in caged mosquito populations and efficiently promoted the simultaneous spread of the antimalarial Lipophorin::Sc2A10 allele. This combination is expected to decrease parasite transmission via two different mechanisms. This work contributes to the design of novel strategies to spread antimalarial transgenes in mosquitoes, and illustrates some expected and unexpected outcomes encountered when establishing a population modification gene drive.
A homing rescue gene drive with multiplexed gRNAs reaches high frequency in cage populations but generates functional resistance
28651Jingheng Chen, Shibo Hou, Ruobing Feng, Xuejiao Xu, Nan Liang, Jackson Champer, bioRxiv, 2023-12-05 10:59:48.
CRISPR homing gene drive is a potent technology with considerable potential for managing populations of medically and agriculturally significant insects. It induces a bias in the inheritance of the drive allele in progeny, rapidly spreading desired genes throughout the population. Homing drives operate by Cas9 cleavage followed by homology-directed repair, copying the drive allele to the wild-type chromosome. However, resistance alleles formed by end-joining repair pose a significant obstacle to the spread of the drive. To address this challenge, we created a homing drive targeting the essential but haplosufficient hairy gene. Our strategy involves spreading the drive construct through the homing process, eliminating nonfunctional resistance, which are recessive lethal, while rescuing drive-carrying individuals with a recoded version of hairy. This strategy eliminates resistance more slowly than a previous strategy targeting haplolethal genes, but it may be easier to construct in non-model organisms. Our drive inheritance rate was moderate, and multigenerational cage studies showed quick drive spread to 96-97% of the population. However, the drive failed to reach the whole population due to the formation of functional resistance alleles, despite use of four gRNAs, a strategy that previously was successful at preventing functional resistance. Sequencing showed that these alleles had a large deletion and must have utilized an alternate start codon. The resistance allele had a modest fitness advantage over the drive in a cage study, which could prevent long-term persistence of the drive, especially if cargo genes had an additional fitness cost. Thus, revised design strategies targeting more essential regions of a target gene may often be necessary to avoid such functional resistance, even when using multiplexed gRNAs.
Invasive Feral Cats Could Be Wiped Out Using Genetic Modification
28632Jess Thomson, Newsweek, 2023-12-04 10:38:32.
Hordes of feral cats terrorizing native species in Australia could be combatted using a special type of genetic engineering, scientists have suggested. The cats, which came to Australia via European colonizers, regularly kill native mammals, birds, and reptiles, including woylies, quolls, and even penguins. The feral cats now number over six million, and are responsible for the extinction of at least 28 species across the country, threatening countless more. This has spurred numerous control measures to be announced, including poison, trapping, and cat curfews. "Gene drives literally 'drive' modified genes through a species by ensuring they are inherited from generation to generation, eventually resulting in the whole species having engineered genetic traits," Andrew D. Maynard, a professor of Advanced Technology Transitions at Arizona State University, told Newsweek. "It's a technique that is specific to species that mate and reproduce sexually, and works by ensuring that engineered genetic traits are inherited by every single offspring resulting from mating."
Manipulating the Destiny of Wild Populations Using CRISPR
28626Raban R, Marshall JM, Hay BA, Akbari OS., Annual Reviews, 57:361-390. 2023-12-04 09:57:57.
Genetic biocontrol aims to suppress or modify populations of species to protect public health, agriculture, and biodiversity. Advancements in genome engineering technologies have fueled a surge in research in this field, with one gene editing technology, CRISPR, leading the charge. This review focuses on the current state of CRISPR technologies for genetic biocontrol of pests and highlights the progress and ongoing challenges of using these approaches.
Gene Drive Mosquitoes from Islamic Perspective: A Preliminary Discussion
28385N. M. Isa, Global Journal Al-Thaqafah, 13. 2023-11-11 10:14:15.
Gene drive mosquitoes could spread desired trait, such as female infertility within a wild population at a rate higher than the normal inheritance rate and could eventually wipe out the population. Consequently, this makes gene drive mosquitoes one of the promising approaches in controlling mosquito-borne diseases, such as malaria and dengue. Despite its potentials, the development of gene drive mosquitoes has raised ethical concerns, mainly on the issues of safety and efficacy, as well as tampering with nature. Little research has been conducted to explore religious perspectives on this new advancement. This article aims to fill that gap by exploring the ethics of gene drive mosquitoes from Islamic perspectives. This article outlines three aspects, namely the purposes, the potential benefits and harms, and the need of the technology that should be considered when discussing whether gene drive mosquitoes should be allowed from Islamic perspectives. © (2023), (Universiti Sultan Azlan Shah). All Rights Reserved.
Generating and testing reagents for CRISPR/Cas9 based homologous recombination and gene drive in Tribolium
28363C. M. Hannah, J. H. Kennedy, M. Megan, E. Z. Gabriel, W. Michael and Z. Andrew, bioRxiv, 2023.11.07.566100. 2023-11-08 09:45:24.
CRISPR/Cas9 gene drive systems are possible in a few insects and ever expanding. Nonetheless, success in one species and techniques developed for it are not necessarily applicable to other species. As such, the development and expansion of gene drive systems is dependent upon direct experimentation. A critical aspect and potentially limiting factor of gene drive is the ability to induce Cas9-dependent homologous recombination. Here we report our attempts to induce Cas9-dependent homologous recombination and subsequent gene drive in Tribolium castaneum. Utilizing constructs containing one or two target gRNAs in combination with Cas9 under two different promoters and corresponding homology arms, we found a high incidence of CRISPR/Cas9 induced mutations but a complete lack of evidence of homologous recombination and genetic drive. Even though the generated constructs provide new resources for CRISPR/Cas9 modification of the Tribolium genome, our results suggest that Tribolium genome may be refractory towards Cas9-induced homologous recombination and additional modifications will be necessary to increase the potential for homologous recombination.Competing Interest StatementThe authors have declared no competing interest.
Overriding Mendelian inheritance in Arabidopsis with a CRISPR toxin-antidote gene drive that impairs pollen germination
28101L. Yang, J. Bingke, C. Jackson and Q. Wenfeng, bioRxiv, 2023.10.10.561637. 2023-10-11 07:52:15.
Synthetic gene drives, inspired by natural selfish genetic elements, present transformative potential for disseminating traits that benefit humans throughout wild populations, irrespective of potential fitness costs. Here, we constructed a gene drive system called CRISPR-Assisted Inheritance utilizing NPG1 (CAIN), which employs a toxin-antidote mechanism in the male germline to override Mendelian inheritance in plants. Specifically, a gRNA-Cas9 cassette targets the essential No Pollen Germination 1 (NPG1) gene, serving as the toxin to block pollen germination. A recoded, CRISPR-resistant copy of NPG1 serves as the antidote, providing rescue only in pollen cells that carry the drive. To limit potential consequences of inadvertent release, we used self-pollinating Arabidopsis thaliana as a model. The drive demonstrated a robust 88-99% transmission rate over two successive generations, producing minimal resistance alleles that are unlikely to inhibit drive spread. Our study provides a strong basis for rapid genetic modification or suppression of outcrossing plant populations.Competing Interest StatementThe authors have declared no competing interest.
MGDrivE 3: A decoupled vector-human framework for epidemiological simulation of mosquito genetic control tools and their surveillance
27894A. Mondal, C. H. M. Sanchez and J. M. Marshall, bioRxiv, 2023.09.09.556958. 2023-09-12 08:11:56.
We present MGDrivE 3 (Mosquito Gene Drive Explorer 3), a new version of a previously-developed framework, MGDrivE 2, that investigates the spatial population dynamics of mosquito genetic control systems and their epidemiological implications. The new framework incorporates three major developments: i) a decoupled sampling algorithm allowing the vector portion of the MGDrivE framework to be paired with a more detailed epidemiological framework, ii) a version of the Imperial College London malaria transmission model, which incorporates age structure, various forms of immunity, and human and vector interventions, and iii) a surveillance module that tracks mosquitoes captured by traps throughout the simulation. Example MGDrivE 3 simulations are presented demonstrating the application of the framework to a CRISPR-based homing gene drive linked to dual disease-refractory genes and their potential to interrupt local malaria transmission. Simulations are also presented demonstrating surveillance of such a system by a network of mosquito traps. MGDrivE 3 is freely available as an open-source R package on CRAN (https://cran.r-project.org/package=MGDrivE2) (version 2.1.0), and extensive examples and vignettes are provided.
A migration-selection model in genetic engineering
27690Y. Qi and L. Su, Nonlinear Analysis: Real World Applications, 75:103983. 2023-08-31 08:48:22.
We investigate a migration-selection system arising from CRISPR-Cas9 genetic engineering, which describes the evolution of the frequencies of a wild allele O, a drive allele D, and a brake allele B. The purpose is to see whether the drive allele D can persist in the population and whether its spread can be limited or stopped by the brake allele B when necessary. We give a complete classification of the dynamics of this system when there is no migration. We further show that migration may cause complex spatiotemporal patterns by demonstrating the existence of spatially inhomogeneous periodic solutions and steady state solutions.
Gene drives for invasive wasp control: Extinction is unlikely, with suppression dependent on dispersal and growth rates
27669P. J. Lester, D. O'Sullivan and G. L. W. Perry, Ecological Applications, 2023-08-24 06:36:30.
Abstract Gene drives offer a potentially revolutionary method for pest control over large spatial extents. These genetic modifications spread deleterious variants through a population and have been proposed as methods for pest suppression or even eradication. We examined the influence of local dispersal, long-distance and/or human-mediated dispersal, and variation in population growth, on the success of a gene drive for the control of invasive social wasps (Vespula vulgaris). Our simulations incorporated a spatially realistic environment containing variable habitat quality in New Zealand. Pest eradication was not observed, except in extreme and unrealistic scenarios of constant, widespread, and spatially intense releases of genetically modified individuals every year for decades. Instead, the regional persistence of genetically modified and wild-type wasps was predicted. Simulations using spatially homogeneous versus realistic landscapes (incorporating uninhabitable areas and dispersal barriers) showed little difference in overall population dynamics. Overall, little impact on wasp abundance was observed in the first 15?years post-introduction. After 25?years, populations were suppressed to levels <95% of starting populations. Populations exhibited ?chase dynamics? with population cycles in space, with local extinction occurring in some areas while wasps became abundant in others. Increasing the wasps' local dispersal distance increased the spatial and temporal variability of the occupied area and population suppression. Varying levels of human-associated long-distance dispersal had little effect on population dynamics. Increasing intrinsic population growth rates interacted with local dispersal to cause higher mean populations and substantially higher levels of variation in population suppression and the total amount of landscape occupied. Gene drives appear unlikely to cause a rapid and widespread extinction of this and probably other pests, but could offer long-term and cost-effective methods of pest suppression. The predicted level of <95% pest suppression would substantially reduce the predation pressure and competitive interactions of this invasive wasp on native species. However, the predicted long-term persistence of genetically modified pests will influence the ethics and likelihood of using gene drives for pest control, especially given concerns that modified wasps would eventually be transported back to their home range.
Population suppression by release of insects carrying a dominant sterile homing gene drive targeting doublesex in Drosophila
26903C. Weizhe, G. Jialiang, L. Yiran and C. Jackson, bioRxiv, 2023.07.17.549342. 2023-07-17 11:00:13.
Gene drive alleles, which bias their own inheritance and increase in frequency, show great promise for blocking disease transmission or directly suppressing pest populations. The most common engineered drive system is the CRISPR homing drive, which converts wild-type alleles to drive alleles in the germline of drive heterozygotes by homology-directed repair after CRISPR cleavage. One successful homing drive example targets a female-specific exon in doublesex in Anopheles mosquitos, suppressing the population by inducing recessive sterility in female drive homozygotes. We found that in Drosophila melanogaster, a 3-gRNA drive disrupting the doublesex female exon resulted in a masculine phenotype and dominant female sterility. Resistance alleles formed by end-joining repair were also dominant sterile. This was likely caused by expression of male-specific transcripts in females with drive and resistance alleles, disrupting sex development. Based on this construct, we proposed a new pest suppression system called Release of Insects carrying a Dominant-sterile Drive (RIDD). This entails continuously releasing drive heterozygous males, with drive and resistance alleles causing sterility in females. The drive remains at high frequency longer than currently used dominant female-lethal alleles (RIDL) due to drive conversion in males, and drive alleles also cause sterility based on resistance, both substantial advantages. With weekly releases of drive males into a cage population with overlapping generations, our RIDD system targeting dsx reached 100% prevalence within 27 weeks, progressively reducing egg production and eventually causing total population collapse. RIDD combines the merits of homing gene drive and RIDL. It is powerful but self-limiting, unlike unconfined standard homing drives, allowing for targeted population suppression.Competing Interest StatementThe authors have declared no competing interest.
New germline Cas9 promoters show improved performance for homing gene drive
26623D. Jie, C. Weizhe, J. Xihua, X. Xuejiao, Y. Emily, Z. Ruizhi, Z. Yuqi, M. Matt, W. M. Philipp and C. Jackson, bioRxiv, 2023.07.16.549205. 2023-07-16 08:48:12.
Gene drive systems could be a viable strategy to prevent pathogen transmission or suppress vector populations by propagating drive alleles with super-Mendelian inheritance. CRISPR-based homing gene drives, perhaps the most powerful gene drive strategy, convert wild type alleles into drive alleles in heterozygotes with the help of Cas9 and gRNA. However, achieving successful outcomes with these drives often requires high performance. Specifically, it is desirable to identify Cas9 promoters that yield high drive conversion rates, minimize the formation rate of resistance alleles in both the germline and the early embryo, and limit somatic Cas9 expression. Thus far, high-performance promoters have only been discovered in Anopheles species. In Drosophila, the nanos promoter avoids leaky somatic expression, but at the cost of high embryo resistance from maternally deposited Cas9. To improve drive efficiency, we tested eleven Drosophila melanogaster germline promoters in several configurations. Some of the new promoters achieved higher drive conversion efficiency with minimal embryo resistance, but none could completely avoid somatic expression like nanos. However, such somatic expression often did not carry detectable fitness costs when the promoter-Cas9 elements supported a rescue homing drive targeting a haplolethal gene, suggesting somatic drive conversion. Based on our findings, we selected two Cas9 promoter lines for cage experiments with a 4-gRNA suppression drive. While one promoter exhibited substantial somatic effects, leading to a low drive equilibrium frequency, the other outperformed nanos, resulting in the successful suppression of the cage population. Overall, these novel Cas9 promoters hold potential advantages for homing drives in Drosophila species and may also possess valuable homologs in other organisms.Competing Interest StatementThe authors have declared no competing interest.
CRISPR-based gene drives generate super-Mendelian inheritance in the disease vector Culex quinquefasciatus
25865T. Harvey-Samuel, X. Feng, E. M. Okamoto, D.-K. Purusothaman, P. T. Leftwich, L. Alphey and V. M. Gantz, bioRxiv, 2023.06.12.544656. 2023-06-15 08:40:13.
Culex mosquitoes pose a significant public health threat as vectors for a variety of diseases including West Nile virus and lymphatic filariasis, and transmit pathogens threatening livestock, companion animals, and endangered birds. Rampant insecticide resistance makes controlling these mosquitoes challenging and necessitates the development of new control strategies. Gene drive technologies have made significant progress in other mosquito species, although similar advances have been lagging in Culex. Here we test the first CRISPR-based homing gene drive for Culex quinquefasciatus, demonstrating the possibility of using this technology to control Culex mosquitoes. Our results show that the inheritance of two split-gene-drive transgenes, targeting different loci, are biased in the presence of a Cas9-expressing transgene although with modest efficiencies. Our findings extend the list of disease vectors where engineered homing gene drives have been demonstrated to include Culex alongside Anopheles and Aedes, and pave the way for future development of these technologies to control Culex mosquitoes
CRISPR/Cas9-based split homing gene drive targeting doublesex for population suppression of the global fruit pest Drosophila suzukii
25710A. K. Yadav, C. Butler, A. Yamamoto, A. A. Patil, A. L. Lloyd and M. J. Scott, Proc Natl Acad Sci U S A, 120:e2301525120. 2023-06-13 10:38:33.
Genetic-based methods offer environmentally friendly species-specific approaches for control of insect pests. One method, CRISPR homing gene drive that target genes essential for development, could provide very efficient and cost-effective control. While significant progress has been made in developing homing gene drives for mosquito disease vectors, little progress has been made with agricultural insect pests. Here, we report the development and evaluation of split homing drives that target the doublesex (dsx) gene in Drosophila suzukii, an invasive pest of soft-skinned fruits. The drive component, consisting of dsx single guide RNA and DsRed genes, was introduced into the female-specific exon of dsx, which is essential for function in females but not males. However, in most strains, hemizygous females were sterile and produced the male dsx transcript. With a modified homing drive that included an optimal splice acceptor site, hemizygous females from each of the four independent lines were fertile. High transmission rates of the DsRed gene (94 to 99%) were observed with a line that expressed Cas9 with two nuclear localization sequences from the D. suzukii nanos promoter. Mutant alleles of dsx with small in-frame deletions near the Cas9 cut site were not functional and thus would not provide resistance to drive. Finally, mathematical modeling showed that the strains could be used for suppression of lab cage populations of D. suzukii with repeated releases at relatively low release ratios (1:4). Our results indicate that the split CRISPR homing gene drive strains could potentially provide an effective means for control of D. suzukii populations.
CRISPR/Cas9-based gene drive could suppress agricultural pests
25706North Carolina State University, Phys Org, 2023-06-12 10:22:37.
Researchers have developed a "homing gene drive system" based on CRISPR/Cas9 that could be used to suppress populations of Drosophila suzukii vinegar flies—so-called "spotted-wing Drosophila" that devastate soft-skinned fruit in North America, Europe and parts of South America—according to new research from North Carolina State University. The NC State researchers developed dual CRISPR gene drive systems that targeted a specific D. suzukii gene called doublesex, which is important for sexual development in the flies. CRISPR stands for "clustered regularly interspaced short palindromic repeats" and Cas9 is an enzyme that performs like molecular scissors to cut DNA. CRISPR systems are derived from bacterial immune systems that recognize and destroy viruses and other invaders, and are being developed as solutions to problems in human, plant and animal health, among other uses. Targeting the doublesex gene resulted in female sterility in numerous experiments as females were unable to lay eggs, says Max Scott, an NC State entomologist who is the corresponding author of a paper in Proceedings of the National Academy of Sciences that describes the research. "This is the first so-called homing gene drive in an agricultural pest that potentially could be used for suppression," Scott said. Gene drives can preferentially select, change or delete particular traits or characteristics and "drive" those edits through future generations, resulting in a sometimes far greater than 50% chance of passing those changes to progeny.
Rapid, scalable, combinatorial genome engineering by marker-less enrichment and recombination of genetically engineered loci in yeast
26189M. Abdullah, B. M. Greco, J. M. Laurent, R. K. Garge, D. R. Boutz, M. Vandeloo, E. M. Marcotte and A. H. Kachroo, Cell Rep Methods, 3:100464. 2023-05-10 12:35:58.
A major challenge to rationally building multi-gene processes in yeast arises due to the combinatorics of combining all of the individual edits into the same strain. Here, we present a precise and multi-site genome editing approach that combines all edits without selection markers using CRISPR-Cas9. We demonstrate a highly efficient gene drive that selectively eliminates specific loci by integrating CRISPR-Cas9-mediated double-strand break (DSB) generation and homology-directed recombination with yeast sexual assortment. The method enables marker-less enrichment and recombination of genetically engineered loci (MERGE). We show that MERGE converts single heterologous loci to homozygous loci at ∼100% efficiency, independent of chromosomal location. Furthermore, MERGE is equally efficient at converting and combining multiple loci, thus identifying compatible genotypes. Finally, we establish MERGE proficiency by engineering a fungal carotenoid biosynthesis pathway and most of the human α-proteasome core into yeast. Therefore, MERGE lays the foundation for scalable, combinatorial genome editing in yeast.
Anti-CRISPR Anopheles mosquitoes inhibit gene drive spread under challenging behavioural conditions in large cages
25086A. Simoni, R. D'Amato, C. Taxiarchi, M. Galardini, A. Trusso, R. Minuz, S. Gilli, A. Somerville, D. Shittu, A. Khalil, R. Galizi and R. Muller, Research Square, 2023-04-24 06:46:30.
CRISPR-based gene drives have the potential to spread within a population and are considered as promising vector control tools. A doublesex-targeting gene drive was shown effective to suppress laboratory populations in both small and large cages, and it is considered for field application. Challenges related to the field-use of gene drives and the evolving regulatory framework demand for systems able to modulate or revert the action of gene drives, as part of post-release risk-mitigation plans. We developed an improved AcrIIA4-based anti-drive strain and showed inhibition of gene drive spread, in complex feeding and reproductive behavioural conditions. A stochastic model predicted the experimentally-observed genotypes dynamics in overlapping generations in medium- and large-sized cages and further demonstrated the effectiveness of anti-drive in different release and fitness scenarios. This study provides a further validation for the use of anti-drive system in controlling the spread of gene drive in Anopheles under complex behavioural conditions.
Synthetic gene drives as an anthropogenic evolutionary force
24895A. D. Cutter, Trends in Genetics, 2023-03-28 07:24:22.
Genetic drive represents a fundamental evolutionary force that can exact profound change to the genetic composition of populations by biasing allele transmission. Herein I propose that the use of synthetic homing gene drives, the human-mediated analog of endogenous genetic drives, warrants the designation of ‘genetic welding’ as an anthropogenic evolutionary force. Conceptually, this distinction parallels that of artificial and natural selection. Genetic welding is capable of imposing complex and rapid heritable phenotypic change on entire populations, whether motivated by biodiversity conservation or public health. Unanticipated possible long-term evolutionary outcomes, however, demand further investigation and bioethical consideration. The emerging importance of genetic welding also compels our explicit recognition of genetic drive as an addition to the other four fundamental forces of evolution.
Identification and functional analysis of Cochliomyia hominivorax U6 gene promoters
27884R. Novas, T. Basika, M. E. Williamson, P. Fresia, A. Menchaca and M. J. Scott, Insect Molecular Biology, 2023-03-21 08:32:32.
The New World screwworm, Cochliomyia hominivorax, is an obligate parasite, which is a major pest of livestock. While the sterile insect technique was used very successfully to eradicate C. hominivorax from North and Central America, more cost-effective genetic methods will likely be needed in South America. The recent development of CRISPR/Cas9-based genetic approaches, such as homing gene drive, could provide a very efficient means for the suppression of C. hominivorax populations. One component of a drive system is the guide RNA(s) driven by a U6 gene promoter. Here, we have developed an in vivo assay to evaluate the activity of the promoters from seven C. hominivorax U6 genes. Embryos from the related blowfly Lucilia cuprina were injected with plasmid DNA containing a U6-promoter-guide RNA construct and a source of Cas9, either protein or plasmid DNA. Activity was assessed by the number of site-specific mutations in the targeted gene in hatched larvae. One promoter, Chom U6_b, showed the highest activity. These U6 gene promoters could be used to build CRISPR/Cas9-based genetic systems for the control of C. hominivorax.
CRISPR-based genetic control strategies for insect pests
24978Y. Yan, R. A. Aumann, I. Hacker and M. F. Schetelig, Journal of Integrative Agriculture, 22:651-668. 2023-03-11 07:53:08.
Genetic control strategies such as the sterile insect technique have successfully fought insect pests worldwide. The CRISPR (clustered regularly interspaced short palindromic repeats) technology, together with high-quality genomic resources obtained in more and more species, greatly facilitates the development of novel genetic control insect strains that can be used in area-wide and species-specific pest control programs. Here, we review the research progress towards state-of-art CRISPR-based genetic control strategies, including gene drive, sex ratio distortion, CRISPR-engineered genetic sexing strains, and precision-guided sterile insect technique. These strategies' working mechanisms, potential resistance development mechanisms, and regulations are illustrated and discussed. In addition, recent developments such as stacked and conditional systems are introduced. We envision that the advances in genetic technology will continue to be one of the driving forces for developing the next generation of pest control strategies.
A toxin-antidote CRISPR gene drive system for regional population modification
24826J. Champer, E. Lee, E. Yang, C. Liu, A. G. Clark and P. W. Messer, Nature Communications, 11:1082. 2023-02-27 10:39:13.
Engineered gene drives based on a homing mechanism could rapidly spread genetic alterations through a population. However, such drives face a major obstacle in the form of resistance against the drive. In addition, they are expected to be highly invasive. Here, we introduce the Toxin-Antidote Recessive Embryo (TARE) drive. It functions by disrupting a target gene, forming recessive lethal alleles, while rescuing drive-carrying individuals with a recoded version of the target. Modeling shows that such drives will have threshold-dependent invasion dynamics, spreading only when introduced above a fitness-dependent frequency. We demonstrate a TARE drive in Drosophila with 88-95% transmission by female heterozygotes. This drive was able to spread through a large cage population in just six generations following introduction at 24% frequency without any apparent evolution of resistance. Our results suggest that TARE drives constitute promising candidates for the development of effective, flexible, and regionally confinable drives for population modification.
Simulations Reveal High Efficiency and Confinement of a Population Suppression CRISPR Toxin-Antidote Gene Drive
24791Y. Zhu and J. Champer, ACS Synthetic Biolog, 2023-02-24 09:56:44.
Though engineered gene drives hold great promise for spreading through and suppressing populations of disease vectors or invasive species, complications such as resistance alleles and spatial population structure can prevent their success. Additionally, most forms of suppression drives, such as homing drives or driving Y chromosomes, will generally spread uncontrollably between populations with even small levels of migration. The previously proposed CRISPR-based toxin-antidote system called toxin-antidote dominant embryo (TADE) suppression drive could potentially address the issues of confinement and resistance. However, it is a relatively weak form of drive compared to homing drives, which might make it particularly vulnerable to spatial population structure. In this study, we investigate TADE suppression drive using individual-based simulations in a continuous spatial landscape. We find that the drive is actually more confined than in simple models without space, even in its most efficient form with low cleavage rate in embryos from maternally deposited Cas9. Furthermore, the drive performed well in continuous space scenarios if the initial release requirements were met, suppressing the population in a timely manner without being severely affected by chasing, a phenomenon in which wild-type individuals avoid the drive by recolonizing empty areas. At higher embryo cut rates, the drive loses its ability to spread, but a single, widespread release can often still induce rapid population collapse. Thus, if TADE suppression gene drives can be successfully constructed, they may play an important role in control of disease vectors and invasive species when stringent confinement to target populations is desired.
Gene Drive: Past, Present and Future Roads to Vertebrate Biocontrol
24780G. R. McFarlane, C. B. A. Whitelaw and S. G. Lillico, Applied Biosciences, 2:52-70. 2023-02-13 09:35:36.
Scientists have long sought a technology to humanely control populations of damaging invasive pests in a species-specific manner. Gene drive technology could see this become a reality. This review charts the twists and turns on the road to developing gene drives in vertebrates. We focus on rodents, as these will likely be the first targets, and trace the journey from the early understanding of selfish genetic elements to engineering gene drives in mice; before discussing future research focuses and the crucial role that public perception and governance will play in the application of this technology. The realisation of robust gene drive strategies in vertebrate pests has the potential to revolutionise biocontrol.
Engineered Antiviral Sensor Targets Infected Mosquitoes
24617E. Dalla Benetta, A. J. Lopez-Denman, H.-H. Li, R. A. Masri, D. J. Brogan, M. Bui, T. Yang, M. Li, M. Dunn, M. J. Klein, S. Jackson, K. Catalan, K. R. Blasdell, P. Tng, I. Antoshechkin, L. S. Alphey, P. N. Paradkar and O. Akbari, bioRxiv, 2023.01.27.525922. 2023-01-27 09:22:47.
Escalating vector disease burdens pose significant global health risks, so innovative tools for targeting mosquitoes are critical. We engineered an antiviral strategy termed REAPER (vRNA Expression Activates Poisonous Effector Ribonuclease) that leverages the programmable RNA-targeting capabilities of CRISPR Cas13 and its potent collateral activity. Akin to a stealthy Trojan Horse hiding in stealth awaiting the presence of its enemy, REAPER remains concealed within the mosquito until an infectious blood meal is up taken. Upon target viral RNA infection, REAPER activates, triggering programmed destruction of its target arbovirus such as chikungunya. Consequently, Cas13 mediated RNA targeting significantly reduces viral replication and its promiscuous collateral activity can even kill infected mosquitoes. This innovative REAPER technology adds to an arsenal of effective molecular genetic tools to combat mosquito virus transmission.Competing Interest StatementO.S.A is a founder of both Agragene, Inc. and Synvect, Inc. with equity interest. The terms of this arrangement have been reviewed and approved by the University of California, San Diego in accordance with its conflict of interest policies. L.A is an adviser to Synvect, Inc and Biocentis Ltd., with financial interest in each. All other authors declare no competing interests.
Researchers Create New System for Safer Gene-Drive Testing and Development
24471M. Aguilera, UC San Diego Today, 2023-01-12 08:46:16.
In the journal Nature Communications, University of California San Diego researchers led by former Postdoctoral Scholar Gerard Terradas together with Postdoctoral Scholar Zhiqian Li and Professor Ethan Bier, in close collaboration with UC Berkeley graduate student Jared Bennett and Associate Professor John Marshall, describe the development of a new system for testing and developing gene drives in the laboratory and safely converting them into tools for potential real-world applications. “These studies both empower new engineering of gene-drive systems while providing important information regarding how to assess and analyze key interactions between their most important moving parts,” said Bier, a faculty member in the School of Biological Sciences, Department of Cell and Developmental Biology. CRISPR-based gene drives feature a protein called a Cas9 endonuclease and a guide RNA molecule that join forces to direct DNA cuts to specific sites in the genome where new genetic elements can be inserted. As the DNA repairs these cuts, the new genetic elements are copied from one chromosome to another, resulting in offspring that exceed the standard 50-50 percent inheritance, instead favoring the newly inserted genetic elements.
Assessment of distant-site rescue elements for CRISPR toxin-antidote gene drives
24363J. Chen, X. Xu and J. Champer, bioRxiv, 2023.01.06.522951. 2023-01-06 09:33:07.
New types of gene drives promise to provide increased flexibility, offering many options for confined modification or suppression of target populations. Among the most promising are CRISPR toxin-antidote gene drives, which disrupt essential wild-type genes by targeting them with Cas9/gRNA, resulting in their removal. This increases the frequency of the drive in the population. All these drives, plus homing modification rescue drives, rely on having an effective rescue element, which consists of a recoded version of the target gene. This rescue element can be at the same site as the target gene, which maximizes the chance of efficient rescue, or at a distant site, which allows some other useful options, such as easily disrupting another essential gene or achieving greater confinement. Previously, we developed a homing rescue drive targeting a haplolethal gene and a toxin-antidote drive targeting an essential but haplosufficient gene. These successful drives had functional rescue elements but suboptimal drive efficiency. Here, we attempted to construct new toxin-antidote drives targeting these genes with a distantsite configuration from three different loci. We found that use of additional gRNAs increased cut rates to nearly 100%. However, all distant-site rescue elements failed for both haplolethal and haplosufficient target genes. Furthermore, one rescue element with a minimally recoded rescue element was used as a template for homology-directed repair for the target gene on a different chromosomal arm, resulting in the formation of functional resistance alleles at high frequency. Together, these results can inform the design of future CRISPR-based toxin-antidote gene drives.Competing Interest StatementThe authors have declared no competing interest.
The Possibilities of Gene Drives for Managing Populations and Controlling Diseases
27641J. Vijay Upadhye, U. N. Shah and B. Mudhol, Salud, Ciencia y Tecnologia, 3. 2023-01-01 07:51:35.
The technical limitations and the use of gene drives to address ecological problems by modifying all populations of wild species remain primarily speculative. Here, we examine the possibility that RNA-guided gene drives based on the CRISPR nuclease Cas9 could be used as an all-encompassing approach for introducing changed features into natural populations over a long period. We outline potential capabilities and possible disadvantages and offer new preventative measures to stem from the propagation of genes and undo genetic modifications. Editing the sexual animal population would significantly benefit both people and the environment. For instance, RNA-guided gene drives may stop the spread of illness, assist farming by correcting bug and weed chemicals and resistance to herbicides, and manage harmful invasive species. However, each prospective use needs to be carefully evaluated due to the likelihood of unfavorable ecological repercussions and the near inevitability of dissemination beyond political boundaries. To investigate the responsible application of this now hypothetical technology, we want serious, inclusive, educated public conversations. © 2023; Los autores.
CRISPR Gene Drives: A Weapon of Mass Destruction?
24336J. Ng, Medium, 2022-12-29 08:40:22.
Gene drives allow scientists to “drive” new genes — and their associated traits — into wildlife populations at unprecedented rates. Here’s a simplified explanation of how gene drives work. In normal sexual reproduction between species with two copies of chromosomes, each gene has a 50% chance of being inherited. However, there are particular DNA sequences called “selfish genes” whose frequency in the genome increases with each generation, even if this doesn’t result in an evolutionary advantage for the offspring. In 2003, biologist Austin Burt proposed a new way to use selfish genes to spread traits more efficiently through a population and ensure that offspring have a 100% probability of inheriting a particular DNA segment.
Genes drive organisms and slippery slopes
24322D. B. Resnik, R. F. Medina, F. Gould, G. Church and J. Kuzma, Pathog Glob Health, 2022-12-22 08:58:12.
The bioethical debate about using gene drives to alter or eradicate wild populations has focused mostly on issues concerning short-term risk assessment and management, governance and oversight, and public and community engagement, but has not examined big-picture- 'where is this going?'-questions in great depth. In other areas of bioethical controversy, big-picture questions often enter the public forum via slippery slope arguments. Given the incredible potential of gene drive organisms to alter the Earth's biota, it is somewhat surprising that slippery slope arguments have not played a more prominent role in ethical and policy debates about these emerging technologies. In this article, we examine a type of slippery slope argument against using gene drives to alter or suppress wild pest populations and consider whether it has a role to play in ethical and policy debates. Although we conclude that this argument does not provide compelling reasons for banning the use of gene drives in wild pest populations, we believe that it still has value as a morally instructive cautionary narrative that can motivate scientists, ethicists, and members of the public to think more clearly about appropriate vs. inappropriate uses of gene drive technologies, the long-term and cumulative and emergent risks of using gene drives in wild populations, and steps that can be taken to manage these risks, such as protecting wilderness areas where people can enjoy life forms that have not been genetically engineered.
Gene editing and agrifood systems
24291FAO, FAO, 2022-12-20 09:22:56.
Gene-editing technologies represent a promising new tool for plant and animal breeding in low- and middle-income countries. They enhance precision and efficiency over current breeding methods and could lead to rapid development of improved plant varieties and animal breeds. However, as for any new technology, they have their merits and demerits. There is, as yet, no international consensus regarding if and how gene-edited organisms should be regulated, and whether their release would fall under the regulatory framework of the Cartagena Protocol on Biosafety to the Convention on Biological Diversity. This science- and evidence-based Issue Paper on gene editing and agrifood systems presents a balanced discussion of the most pertinent aspects of gene editing, including the consequences for human hunger, human health, food safety, effects on the environment, animal welfare, socioeconomic impact and distribution of benefits. Intrinsic ethical concerns and issues of governance and regulation are addressed, and the roles of the public and private sectors, alone and in partnership, are summarized. Various scenarios are also presented for how gene editing might be used in the future to help transform agrifood systems.
Performance characteristics allow for confinement of a CRISPR toxin-antidote gene drive designed for population suppression
24272S. Zhang and J. Champer, bioRxiv, 2022.12.13.520356. 2022-12-15 14:08:18.
Gene drives alleles that can bias their own inheritance are a promising way to engineer populations for control of disease vectors, invasive species, and agricultural pests. Recent advancements in the field have yielded successful examples of powerful suppression type drives and confined modification type drives, but developing confined suppression drives has proven more difficult. This is because the necessary power for strong suppression is often incompatible with the characteristics needed for drive confinement. However, one type of CRISPR toxin-antidote drive may be strong enough and confined, the TADE (Toxin-Antidote Dominant Embryo) suppression drive. By disrupting a haplolethal target gene and a haplosufficient female fertility gene, this drive quickly eliminates wild-type alleles and eventually induces population suppression. It has been shown to perform effectively in panmictic populations. However, confinement in spatial scenarios may be substantially different. Here, we use a reaction-diffusion model to assess the performance of TADE suppression drive in continuous space. We measure the drive wave advance speed while varying several performance parameters and find that moderate fitness costs or embryo cutting (from maternally deposited nuclease) can eliminate the drive’s ability to form a wave of advance. We assess the release size required for the drive to propagate, and finally, we investigate migration corridor scenarios. Depending on the corridor size and dispersal, it is often possible for the drive to suppress one population and then persist in the corridor without invading the second population. This prevents re-invasion by wild-type, which may be a particularly desirable outcome in some scenarios. Thus, even imperfect variants of TADE suppression drive may be excellent candidates for confined population suppression.Competing Interest StatementThe authors have declared no competing interest.
Bioinformatic and literature assessment of toxicity and allergenicity of a CRISPR-Cas9 engineered gene drive to control the human malaria mosquito vector Anopheles gambiae
24161A. Qureshi and J. B. Connolly, Malaria Journal, 2022-12-06 07:52:06.
Population suppression gene drive is currently being evaluated, including via environmental risk assessment (ERA), for malaria vector control. One such gene drive involves the dsxFCRISPRh transgene encoding (i) hCas9 endonuclease, (i) T1 guide RNA (gRNA) targeting the doublesex locus, and (iii) DsRed fluorescent marker protein, in genetically modified mosquitoes (GMMs). Problem formulation, the first stage of ERA, for environmental releases of dsxFCRISPRh previously identified nine potential harms to the environment or health that could occur, should expressed products of the transgene cause allergenicity or toxicity. Amino acid sequences of hCas9 and DsRed were interrogated against those of toxins or allergens from NCBI, UniProt, COMPARE and AllergenOnline bioinformatic databases and the gRNA was compared with microRNAs from the miRBase database for potential impacts on gene expression associated with toxicity or allergenicity. PubMed was also searched for any evidence of toxicity or allergenicity of Cas9 or DsRed, or of the donor organisms from which these products were originally derive While Cas9 nuclease activity can be toxic to some cell types in vitro and hCas9 was found to share homology with the prokaryotic toxin VapC, there was no evidence of a risk of toxicity to humans and other animals from hCas9. Although hCas9 did contain an 8-mer epitope found in the latex allergen Hev b 9, the full amino acid sequence of hCas9 was not homologous to any known allergens. Combined with a lack of evidence in the literature of Cas9 allergenicity, this indicated negligible risk to humans of allergenicity from hCas9. No matches were found between the gRNA and microRNAs from either Anopheles or humans. Moreover, potential exposure to dsxFCRISPRh transgenic proteins from environmental releases was assessed as negligible.Bioinformatic and literature assessments found no convincing evidence to suggest that transgenic products expressed from dsxFCRISPRh were allergens or toxins, indicating that environmental releases of this population suppression gene drive for malaria vector control should not result in any increased allergenicity or toxicity in humans or animals. These results should also inform evaluations of other GMMs being developed for vector control and in vivo clinical applications of CRISPR-Cas9.
A CRISPR endonuclease gene drive reveals distinct mechanisms of inheritance bias
24021S. A. N. Verkuijl, E. Gonzalez, M. Li, J. X. D. Ang, N. P. Kandul, M. A. E. Anderson, O. S. Akbari, M. B. Bonsall and L. Alphey, Nature Communications, 13:7145. 2022-11-21 09:45:13.
CRISPR/Cas gene drives can bias transgene inheritance through different mechanisms. Homing drives are designed to replace a wild-type allele with a copy of a drive element on the homologous chromosome. In Aedes aegypti, the sex-determining locus is closely linked to the white gene, which was previously used as a target for a homing drive element (wGDe). Here, through an analysis using this linkage we show that in males inheritance bias of wGDe did not occur by homing, rather through increased propagation of the donor drive element. We test the same wGDe drive element with transgenes expressing Cas9 with germline regulatory elements sds3, bgcn, and nup50. We only find inheritance bias through homing, even with the identical nup50-Cas9 transgene. We propose that DNA repair outcomes may be more context dependent than anticipated and that other previously reported homing drives may, in fact, bias their inheritance through other mechanisms.
CRISPR-Mediated Cassette Exchange (CriMCE): A Method to Introduce and Isolate Precise Marker-Less Edits
24026I. Morianou, A. Crisanti, T. Nolan and A. M. Hammond, The CRISPR Journal, 2022-11-15 09:33:31.
The introduction of small unmarked edits to the genome of insects is essential to study the molecular underpinnings of important biological traits, such as resistance to insecticides and genetic control strategies. Advances in CRISPR genome engineering have made this possible, but prohibitively laborious for most laboratories due to low rates of editing and the lack of a selectable marker. To facilitate the generation and isolation of precise marker-less edits we have developed a two-step method based on CRISPR-mediated cassette exchange (CriMCE) of a marked placeholder for a variant of interest. This strategy can be used to introduce a wider range of potential edits compared with previous approaches while consolidating the workflow. We present proof-of-principle that CriMCE is a powerful tool by engineering three single nucleotide polymorphism variants into the genome of Anopheles gambiae, with 5?41???higher rates of editing than homology-directed repair or prime editing.
Leveraging a natural murine meiotic drive to suppress invasive populations
23835L. Gierus, A. Birand, M. D. Bunting, G. I. Godahewa, S. G. Piltz, K. P. Oh, A. J. Piaggio, D. W. Threadgill, J. Godwin, O. Edwards, P. Cassey, J. V. Ross, T. A. A. Prowse and P. Q. Thomas, Proceedings of the National Academy of Sciences, 119:e2213308119. 2022-11-08 09:36:13.
Invasive rodents are a major cause of environmental damage and biodiversity loss, particularly on islands. Unlike insects, genetic biocontrol strategies including population-suppressing gene drives with biased inheritance have not been developed in mice. Here, we demonstrate a gene drive strategy (t(CRISPR)) that leverages super-Mendelian transmission of the t haplotype to spread inactivating mutations in a haplosufficient female fertility gene (Prl). Using spatially explicit individual-based in silico modeling, we show that t(CRISPR) can eradicate island populations under a range of realistic field-based parameter values. We also engineer transgenic t(CRISPR) mice that, crucially, exhibit biased transmission of the modified t haplotype and Prl mutations at levels our modeling predicts would be sufficient for eradication. This is an example of a feasible gene drive system for invasive alien rodent population control.
Assessing single-locus CRISPR/Cas9-based gene drive variants in the mosquito Aedes aegypti via single generation crosses and modeling
23756W. Reid, A. E. Williams, I. Sanchez-Vargas, J. Lin, R. Juncu, K. E. Olson and A. W. E. Franz, G3 Genes|Genomes|Genetics, 2022-10-17 06:25:18.
Critical to the design of a single-locus autonomous GD is that the selected genomic locus is amenable to both GD and appropriate expression of the antiviral effector. In our study, we used reverse engineering to target two intergenic genomic loci, which had previously shown to be highly permissive for antiviral effector gene expression, and we further investigated the use of three promoters (nanos, β2-tubulin, or zpg) for Cas9 expression. We then quantified the accrual of insertions or deletions (indels) after single generation crossings, measured maternal effects, and assessed fitness costs associated with the various transgenic lines to model the rate of GD fixation. Overall, MGDrivE modeling suggested that when an autonomous GD is placed into an intergenic locus, the GD system will eventually be blocked by the accrual of GD blocking resistance alleles and ultimately be lost in the population. Moreover, while genomic locus and promoter selection were critically important for the initial establishment of the autonomous GD, it was the fitness of the GD line that most strongly influenced the persistence of the GD in the simulated population. As such, we propose that when autonomous CRISPR/Cas9 based GD systems are anchored in an intergenic locus, they temporarily result in a strong population replacement effect, but as GD-blocking indels accrue, the GD becomes exhausted due to the fixation of CRISPR resistance alleles.
Combating Mosquito-Borne Diseases with CRISPR
23701N. Spahich, The Scientist, 2022-10-11 08:06:34.
Female mosquitoes are some of the deadliest organisms in the world due to their ability to spread infectious diseases through a simple bite. Mosquito-borne diseases such as yellow fever, Zika, Dengue fever, and malaria kill millions of humans every year, and there are limited therapeutics for their prevention and treatment. While in college, Omar Akbari worked as a public service intern testing the local mosquito population for human pathogens and eradicating these insects with chemicals. During this experience, he felt dissatisfied with the insecticide-based method of controlling mosquito population and wanted to find a better way to tackle the problem of mosquito-borne disease spread. With a multidisciplinary team in his laboratory at the University of California, San Diego, he now develops tools through genetic engineering techniques such as CRISPR to solve the world’s insect control problems.
Fitness effects of CRISPR endonucleases in Drosophila melanogaster populations
23673A. M. Langmüller, J. Champer, S. Lapinska, L. Xie, M. Metzloff, S. E. Champer, J. Liu, Y. Xu, J. Du, A. G. Clark and P. W. Messer, eLife, 11:e71809. 2022-09-22 08:22:04.
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 provides a highly efficient and flexible genome editing technology with numerous potential applications ranging from gene therapy to population control. Some proposed applications involve the integration of CRISPR/Cas9 endonucleases into an organism’s genome, which raises questions about potentially harmful effects to the transgenic individuals. One example for which this is particularly relevant are CRISPR-based gene drives conceived for the genetic alteration of entire populations. The performance of such drives can strongly depend on fitness costs experienced by drive carriers, yet relatively little is known about the magnitude and causes of these costs. Here, we assess the fitness effects of genomic CRISPR/Cas9 expression in Drosophila melanogaster cage populations by tracking allele frequencies of four different transgenic constructs that allow us to disentangle ‘direct’ fitness costs due to the integration, expression, and target-site activity of Cas9, from fitness costs due to potential off-target cleavage. Using a maximum likelihood framework, we find that a model with no direct fitness costs but moderate costs due to off-target effects fits our cage data best. Consistent with this, we do not observe fitness costs for a construct with Cas9HF1, a high-fidelity version of Cas9. We further demonstrate that using Cas9HF1 instead of standard Cas9 in a homing drive achieves similar drive conversion efficiency. These results suggest that gene drives should be designed with high-fidelity endonucleases and may have implications for other applications that involve genomic integration of CRISPR endonucleases.
A detailed landscape of CRISPR-Cas-mediated plant disease and pest management
23532S. Karmakar, P. Das, D. Panda, K. Xie, M. J. Baig and K. A. Molla, Plant Science, 323:111376. 2022-09-09 05:44:18.
Genome editing technology has rapidly evolved to knock-out genes, create targeted genetic variation, install precise insertion/deletion and single nucleotide changes, and perform large-scale alteration. The flexible and multipurpose editing technologies have started playing a substantial role in the field of plant disease management. CRISPR-Cas has reduced many limitations of earlier technologies and emerged as a versatile toolbox for genome manipulation. This review summarizes the phenomenal progress of the use of the CRISPR toolkit in the field of plant pathology. CRISPR-Cas toolbox aids in the basic studies on host-pathogen interaction, in identifying virulence genes in pathogens, deciphering resistance and susceptibility factors in host plants, and engineering host genome for developing resistance. We extensively reviewed the successful genome editing applications for host plant resistance against a wide range of biotic factors, including viruses, fungi, oomycetes, bacteria, nematodes, insect pests, and parasitic plants. Recent use of CRISPR-Cas gene drive to suppress the population of pathogens and pests has also been discussed. Furthermore, we highlight exciting new uses of the CRISPR-Cas system as diagnostic tools, which rapidly detect pathogenic microorganism. This comprehensive yet concise review discusses innumerable strategies to reduce the burden of crop protection.
Genetic Tools for Integrated Management of Pests on Honeybees in the Tropics
25464M. Pattabhiramaiah, S. Mallikarjunaiah and D. Brueckner, Genetic Methods and Tools for Managing Crop Pests, 2022-08-22 09:48:31.
The Asian honeybee is endemic to Asia where it has been used for honey production and pollination services from time immemorial. They are integral to modern agricultural productivity and to survival and vitality of natural ecosystems. However, recent declines in populations and species diversity threaten both food security and natural habitats. Honeybee colonies are assaulted by numerous pests and pathogens including mites and beetles. Novel, cost-effective pest management practices are desperately needed to preserve colony health. Increased pest pressure levels justify the need for additional control methods. Beekeepers should follow the guidelines of integrated pest management (IPM) as an effective control option that will have a minimum impact on honeybee health. The use of genomic tools and engineering technologies has a great potential for enhancement and sustenance of the health of honeybees. This review focusses on the application of innovative advanced genetic tools such as SIT (sterile insect technique), genome editing, gene drive, RNAi, CRISPR/Cas9-mediated gene editing, and gene pyramiding in honeybee pest management.
A theory of resistance to multiplexed gene drive demonstrates the significant role of weakly deleterious natural genetic variation
23362B. S. Khatri and A. Burt, Proceedings of the National Academy of Sciences, 119:e2200567119. 2022-08-01 07:26:58.
CRISPR-based gene drives have the potential for controlling natural populations of disease vectors, such as malaria-carrying mosquitoes in sub-Saharan Africa. If successful, they hold promise of significantly reducing the burden of disease and death from malaria and many other vector-borne diseases. A significant challenge to success is the evolution of resistance. Here, we develop a theory of resistance for multiplexed drive, which shows the importance of weakly deleterious naturally occurring genetic variation, whose effect is significantly amplified compared to de novo mutation. These results provide a fundamental basis to estimate how many guide RNAs are required to prevent resistance in the face of natural genetic variation. Evolution of resistance is a major barrier to successful deployment of gene-drive systems to suppress natural populations, which could greatly reduce the burden of many vector-borne diseases. Multiplexed guide RNAs (gRNAs) that require resistance mutations in all target cut sites are a promising antiresistance strategy since, in principle, resistance would only arise in unrealistically large populations. Using stochastic simulations that accurately model evolution at very large population sizes, we explore the probability of resistance due to three important mechanisms: 1) nonhomologous end-joining mutations, 2) single-nucleotide mutants arising de novo, or 3) single-nucleotide polymorphisms preexisting as standing variation. Our results explore the relative importance of these mechanisms and highlight a complexity of the mutation?selection?drift balance between haplotypes with complete resistance and those with an incomplete number of resistant alleles. We find that this leads to a phenomenon where weakly deleterious naturally occurring variants greatly amplify the probability of multisite resistance compared to de novo mutation. This key result provides design criterion for antiresistance multiplexed systems, which, in general, will need a larger number of gRNAs compared to de novo expectations. This theory may have wider application to the evolution of resistance or evolutionary rescue when multiple changes are required before selection can act.
Development of CRISPR/Cas9-Mediated Gene-Drive Construct Targeting the Phenotypic Gene in Plutella xylostella
23265M. Asad, D. Liu, J. Li, J. Chen and G. Yang, Frontiers in Physiology, 13:938621. 2022-07-20 13:03:05.
The gene-drive system can ensure that desirable traits are transmitted to the progeny more than the normal Mendelian segregation. The clustered regularly interspersed palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) mediated gene-drive system has been demonstrated in dipteran insect species, including Drosophila and Anopheles, not yet in other insect species. Here, we have developed a single CRISPR/Cas9-mediated gene-drive construct for Plutella xylostella, a highly-destructive lepidopteran pest of cruciferous crops. The gene-drive construct was developed containing a Cas9 gene, a marker gene (EGFP) and a gRNA sequence targeting the phenotypic marker gene (Pxyellow) and site-specifically inserted into the P. xylostella genome. This homing-based gene-drive copied ∼12 kb of a fragment containing Cas9 gene, gRNA, and EGFP gene along with their promoters to the target site. Overall, 6.67%-12.59% gene-drive efficiency due to homology-directed repair (HDR), and 80.93%-86.77% resistant-allele formation due to non-homologous-end joining (NHEJ) were observed. Furthermore, the transgenic progeny derived from male parents showed a higher gene-drive efficiency compared with transgenic progeny derived from female parents. This study demonstrates the feasibility of the CRISPR/Cas9-mediated gene-drive construct in P. xylostella that inherits the desired traits to the progeny. The finding of this study provides a foundation to develop an effective CRISPR/Cas9-mediated gene-drive system for pest control.
CRISPR-Mediated Genome Engineering in Aedes aegypti
23209R. Sun, M. Li, C. J. McMeniman and O. S. Akbari, piRNA: Methods and Protocols, 2022-07-01 06:21:38.
CRISPR-mediated genome engineering technologies have been adapted to a wide variety of organisms with high efficiency and specificity. The yellow fever mosquito, Aedes aegyptiAedes aegypti, is one such organism. It is also responsible for transmitting a wide variety of deadly viruses including Dengue, Zika, Yellow fever, and Chikungunya. The key to successful CRISPR-mediated gene editingGene editing applications is the delivery of both Cas9 ribonuclease and single-guide RNA (sgRNASingle guide RNA (sgRNA)) to the nucleus of desired cells. Various methods have been developed for supplying the Cas9 endonuclease, sgRNASingle guide RNA (sgRNA), and donor DNA to Ae. aegypti. In this chapter, we focus on methods of direct embryoEmbryosdelivery of editing components, presenting detailed step-by-step CRISPR/Cas9-based genome-editing protocols for inducing desired heritable edits in mosquitoes as well as insights into successful application of these protocols. We also highlight potential opportunities for customizing these protocols to manipulate the mosquito genome for innovative in vivoIn vivo gene function studies.
Gene Drives: A Potentially New Weapon Against Mosquitoes
22905M. Sherman, Times Union Online, 2022-06-13 06:36:32.
Scientists have studied gene drives for more than 50 years, and to most of us this has been a well-kept secret. The development of a powerful genome editing tool in 2012, CRISPR/Cas9,1 led to recent breakthroughs in gene drive research that built on that half century’s worth of knowledge, and stimulated new discussions of the potential applications and implications of gene drive technologies. Just prior to the beginning of this study and since the committee was first convened, scientists published four proofs of concept — one in yeast, one in fruit flies, and two in different species of mosquitoes — that demonstrate the successful development of gene drives in the laboratory, at least in these organisms.Proposed applications for gene-drive modified organisms for basic research, conservation, agriculture, public health and other purposes will likely continue to expand as gene editing tools become more refined. Gene-drive modified organisms are on the horizon. With mosquitoes, the gene drive interferes with the insect’s ability to reproduce. It wiped out captive populations in eight or 12 generations. The first experimental release could be rolled out in Burkina Faso, Mali, Ghana or Uganda.
Active genetics comes alive
22888V. M. Gantz and E. Bier, BioEssays, 2022-06-09 09:28:52.
Abstract Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based ?active genetic? elements developed in 2015 bypassed the fundamental rules of traditional genetics. Inherited in a super-Mendelian fashion, such selfish genetic entities offered a variety of potential applications including: gene-drives to disseminate gene cassettes carrying desired traits throughout insect populations to control disease vectors or pest species, allelic drives biasing inheritance of preferred allelic variants, neutralizing genetic elements to delete and replace or to halt the spread of gene-drives, split-drives with the core constituent Cas9 endonuclease and guide RNA (gRNA) components inserted at separate genomic locations to accelerate assembly of complex arrays of genetic traits or to gain genetic entry into novel organisms (vertebrates, plants, bacteria), and interhomolog based copying systems in somatic cells to develop tools for treating inherited or infectious diseases. Here, we summarize the substantial advances that have been made on all of these fronts and look forward to the next phase of this rapidly expanding and impactful field.
Investigating CRISPR/Cas9 gene drive for production of disease-preventing prion gene alleles
22796A. R. Castle, S. Wohlgemuth, L. Arce and D. Westaway, PLoS One, 17:e0269342. 2022-06-07 09:14:53.
Prion diseases are a group of fatal neurodegenerative disorders that includes chronic wasting disease, which affects cervids and is highly transmissible. Given that chronic wasting disease prevalence exceeds 30% in some endemic areas of North America, and that eventual transmission to other mammalian species, potentially including humans, cannot be ruled out, novel control strategies beyond population management via hunting and/or culling must be investigated. Prion diseases depend upon post-translational conversion of the cellular prion protein, encoded by the Prnp gene, into a disease-associated conformation; ablation of cellular prion protein expression, which is generally well-tolerated, eliminates prion disease susceptibility entirely. Inspired by demonstrations of gene drive in caged mosquito species, we aimed to test whether a CRISPR/Cas9-based gene drive mechanism could, in principle, promote the spread of a null Prnp allele among mammalian populations. First, we showed that transient co-expression of Cas9 and Prnp-directed guide RNAs in RK13 cells generates indels within the Prnp open-reading frame, indicating that repair of Cas9-induced double-strand breaks by non-homologous end-joining had taken place. Second, we integrated a ~1.2 kb donor DNA sequence into the Prnp open-reading frame in N2a cells by homology-directed repair following Cas9-induced cleavages and confirmed that integration occurred precisely in most cases. Third, we demonstrated that electroporation of Cas9/guide RNA ribonucleoprotein complexes into fertilised mouse oocytes resulted in pups with a variety of disruptions to the Prnp open reading frame, with a new coisogenic line of Prnp-null mice obtained as part of this work. However, a technical challenge in obtaining expression of Cas9 in the male germline prevented implementation of a complete gene drive mechanism in mice.
Gene Editing and Genetic Control of Hemipteran Pests: Progress, Challenges and Perspectives
22757I. D. Pacheco, L. L. Walling and P. W. Atkinson, Frontiers in Bioengineering and Biotechnology, 10. 2022-06-07 08:02:11.
The origin of the order Hemiptera can be traced to the late Permian Period more than 230 MYA, well before the origin of flowering plants 100 MY later in during the Cretaceous period. Hemipteran species consume their liquid diets using a sucking proboscis; for phytophagous hemipterans their mouthparts (stylets) are elegant structures that enable voracious feeding from plant xylem or phloem. This adaptation has resulted in some hemipteran species becoming globally significant pests of agriculture resulting in significant annual crop losses. Due to the reliance on chemical insecticides for the control of insect pests in agricultural settings, many hemipteran pests have evolved resistance to insecticides resulting in an urgent need to develop new, species-specific and environmentally friendly methods of pest control. The rapid advances in CRISPR/Cas9 technologies in model insects such as Drosophila melanogaster, Tribolium castaneum, Bombyx mori, and Aedes aegypti has spurred a new round of innovative genetic control strategies in the Diptera and Lepidoptera and an increased interest in assessing genetic control technologies for the Hemiptera. Genetic control approaches in the Hemiptera have, to date, been largely overlooked due to the problems of introducing genetic material into the germline of these insects. The high frequency of CRISPR-mediated mutagenesis in model insect species suggest that, if the delivery problem for Hemiptera could be solved, then gene editing in the Hemiptera might be quickly achieved. Significant advances in CRISPR/Cas9 editing have been realized in nine species of Hemiptera over the past 4 years. Here we review progress in the Hemiptera and discuss the challenges and opportunities for extending contemporary genetic control strategies into species in this agriculturally important insect orderr.
A nickase Cas9 gene-drive system promotes super-Mendelian inheritance in Drosophila
22584V. L. Del Amo, S. S. Juste and V. M. Gantz, Cell Rep, 39:110843. 2022-05-24 09:32:51.
CRISPR-based gene-drives have been proposed for managing insect populations, including disease-transmitting mosquitoes, due to their ability to bias their inheritance toward super-Mendelian rates (>50%). Current technologies use a Cas9 that introduces DNA double-strand breaks into the opposing wild-type allele to replace it with a copy of the gene-drive allele via DNA homology-directed repair. However, the use of different Cas9 versions is unexplored, and alternative approaches could increase the available toolkit for gene-drive designs. Here, we report a gene-drive that relies on Cas9 nickases that generate staggered paired nicks in DNA to propagate the engineered gene-drive cassette. We show that generating 5' overhangs in the system yields efficient allelic conversion. The nickase gene-drive arrangement produces large, stereotyped deletions that are advantageous to eliminate viable animals carrying small mutations when targeting essential genes. Our nickase approach should expand the repertoire for gene-drive arrangements aimed at applications in mosquitoes and beyond.
Intronic gRNAs for the Construction of Minimal Gene Drive Systems
22478A. Nash, P. Capriotti, A. Hoermann, P. A. Papathanos and N. Windbichler, Frontiers in Bioengineering and Biotechnology, 10. 2022-05-12 07:00:08.
Gene drives are promising tools for the genetic control of insect vector or pest populations. CRISPR-based gene drives are generally highly complex synthetic constructs consisting of multiple transgenes and their respective regulatory elements. This complicates the generation of new gene drives and the testing of the behavior of their constituent functional modules. Here, we explored the minimal genetic components needed to constitute autonomous gene drives in Drosophila melanogaster. We first designed intronic gRNAs that can be located directly within coding transgene sequences and tested their functions in cell lines. We then integrated a Cas9 open reading frame hosting such an intronic gRNA within the Drosophila rcd-1r locus that drives the expression in the male and female germlines. We showed that upon removal of the fluorescent transformation marker, the rcd-1rd allele supports efficient gene drive. We assessed the propensity of this driver, designed to be neutral with regards to fitness and host gene function, to propagate in caged fly populations. Because of their simplicity, such integral gene drives could enable the modularization of drive and effector functions. We also discussed the possible biosafety implications of minimal and possibly recoded gene drives.
Double-tap gene drive uses iterative genome targeting to help overcome resistance alleles
22286A. L. Bishop, V. López Del Amo, E. M. Okamoto, Z. Bodai, A. C. Komor and V. M. Gantz, Nat Commun, 13:2595. 2022-05-09 08:10:07.
Homing CRISPR gene drives could aid in curbing the spread of vector-borne diseases and controlling crop pest and invasive species populations due to an inheritance rate that surpasses Mendelian laws. However, this technology suffers from resistance alleles formed when the drive-induced DNA break is repaired by error-prone pathways, which creates mutations that disrupt the gRNA recognition sequence and prevent further gene-drive propagation. Here, we attempt to counteract this by encoding additional gRNAs that target the most commonly generated resistance alleles into the gene drive, allowing a second opportunity at gene-drive conversion. Our presented "double-tap" strategy improved drive efficiency by recycling resistance alleles. The double-tap drive also efficiently spreads in caged populations, outperforming the control drive. Overall, this double-tap strategy can be readily implemented in any CRISPR-based gene drive to improve performance, and similar approaches could benefit other systems suffering from low HDR frequencies, such as mammalian cells or mouse germline transformations.
CRISPR/Cas9 mediates efficient site-specific mutagenesis of the odorant receptor co-receptor (Orco) in the malaria vector Anopheles sinensis
22554Y. Wang, X. F. He, L. Qiao, Z. R. Yu, B. Chen and Z. B. He, Pest Management Science, 11. 2022-04-28 14:52:27.
BACKGROUND Anopheles sinensis is the most widely distributed mosquito species and is the main transmitter of Plasmodium vivax malaria in China. Most previous research has focused on the mechanistic understanding of biological processes in An. sinensis and novel ways of interrupting malaria transmission. However, the development of functional genomics and genetics-based vector control strategies against An. sinensis remain limited because of insufficient site-specific genome editing tools. RESULTS We report the first successful application of the CRISPR/Cas9 mediated knock-in for highly efficient, site-specific mutagenesis in An. sinensis. The EGFP marker gene driven by the 3 x P3 promoter was precisely integrated into the odorant receptor co-receptor (Orco) by direct injections of Cas9 protein, double-stranded DNA donor, and Orco-gRNA. We achieved a mutation rate of 3.77%, similar to rates in other mosquito species. Precise knock-in at the intended locus was confirmed by polymerase chain reaction (PCR) amplification and sequencing. The Orco mutation severely impaired mosquito sensitivity to some odors and their ability to locate and discriminate a human host. CONCLUSION Orco was confirmed as a key mediator of multiple olfactory-driven behaviors in the An. sinensis life cycle, highlighting the importance of Orco as a key molecular target for malaria control. The results also demonstrated that CRISPR/Cas9 was a simple and highly efficient genome editing technique for An. sinensis and could be used to develop genetic control tools for this vector. (c) 2022 Society of Chemical Industry.
A New Approach to Develop Resistant Cultivars Against the Plant Pathogens: CRISPR Drives
21915M. I. Tek and K. Budak, Frontiers in Plant Science, 13. 2022-04-27 08:43:50.
CRISPR drive is a recent and robust tool that allows durable genetic manipulation of the pest population like human disease vectors such as malaria vector mosquitos. In recent years, it has been suggested that CRISPR drives can also be used to control plant diseases, pests, and weeds. However, using a CRISPR drive in Arabidopsis for the first time in 2021 has been shown to use this technology in plant breeding to obtain homozygous parental lines. This perspective has proposed using CRISPR drive to develop pathogen-resistant cultivars by disrupting the susceptibility gene (S). In the breeding program, CRISPR is used to create S-gene mutations in two parental lines of hybrid cultivars. However, CRISPR must be reapplied or long-term backcrossed for the parental line to obtain homozygous S-mutant cultivars. When a parental line crosses with different parental lines to develop new hybrids, heterozygous S-mutations could not resist in hybrid against the pathogen. CRISPR drives are theoretically valid to develop homozygous S-mutant plants against pathogens by only routine pollination after CRISPR drive transformation to just one parental line. This way, breeders could use this parental line in different crossing combinations without reapplying the genome-editing technique or backcrossing. Moreover, CRISPR drive also could allow the development of marker-free resistant cultivars with modifications on the drive cassette.
Biotechnological Road Map for Innovative Weed Management
22450A. C. S. Wong, K. Massel, Y. Lam, J. Hintzsche and B. S. Chauhan, Frontiers in Plant Science, 13. 2022-04-25 09:27:40.
In most agriculture farmlands, weed management is predominantly reliant on integrated weed management (IWM) strategies, such as herbicide application. However, the overuse and misuse of herbicides, coupled with the lack of novel active ingredients, has resulted in the uptrend of herbicide-resistant weeds globally. Moreover, weedy traits that contribute to weed seed bank persistence further exacerbate the challenges in weed management. Despite ongoing efforts in identifying and improving current weed management processes, the pressing need for novel control techniques in agricultural weed management should not be overlooked. The advent of CRISPR/Cas9 gene-editing systems, coupled with the recent advances in “omics” and cheaper sequencing technologies, has brought into focus the potential of managing weeds in farmlands through direct genetic control approaches, but could be achieved stably or transiently. These approaches encompass a range of technologies that could potentially manipulate expression of key genes in weeds to reduce its fitness and competitiveness, or, by altering the crop to improve its competitiveness or herbicide tolerance. The push for reducing or circumventing the use of chemicals in farmlands has provided an added incentive to develop practical and feasible molecular approaches for weed management, although there are significant technical, practical, and regulatory challenges for utilizing these prospective molecular technologies in weed management.
Explainer: The Gene Drive Technology
21701P. Shah, CRISPR Medicine News, 2022-04-11 08:45:54.
Gene drives are genetic elements that can quickly spread through populations and have nearly a 100% chance of passing the genes they carry to the next generation. Synthetic gene drive is a technology of genetic engineering through which certain desired traits can be introduced to almost all individuals in a population. Researchers can either eliminate a species or alter the genetic makeup of living organisms through gene drive technology.
A homing suppression gene drive with multiplexed gRNAs maintains high drive conversion efficiency and avoids functional resistance alleles
22572E. Yang, M. Metzloff, A. M. Langmuller, X. J. Xu, A. G. Clark, P. W. Messer and J. Champer, G3-Genes Genomes Genetics, 13. 2022-04-08 09:02:04.
Gene drives are engineered alleles that can bias inheritance in their favor, allowing them to spread throughout a population. They could potentially be used to modify or suppress pest populations, such as mosquitoes that spread diseases. CRISPR/Cas9 homing drives, which copy themselves by homology-directed repair in drive/wild-type heterozygotes, are a powerful form of gene drive, but they are vulnerable to resistance alleles that preserve the function of their target gene. Such resistance alleles can prevent successful population suppression. Here, we constructed a homing suppression drive in Drosophila melanogaster that utilized multiplexed gRNAs to inhibit the formation of functional resistance alleles in its female fertility target gene. The selected gRNA target sites were close together, preventing reduction in drive conversion efficiency. The construct reached a moderate equilibrium frequency in cage populations without apparent formation of resistance alleles. However, a moderate fitness cost prevented elimination of the cage population, showing the importance of using highly efficient drives in a suppression strategy, even if resistance can be addressed. Nevertheless, our results experimentally demonstrate the viability of the multiplexed gRNAs strategy in homing suppression gene drives.
Cas9-mediated maternal-effect and derived resistance alleles in a gene-drive strain of the African malaria vector mosquito, Anopheles gambiae
21663R. Carballar-Lejarazú, T. Tushar, T. B. Pham and A. A. James, Genetics, 2022-04-07 15:16:08.
CRISPR/Cas9 technologies are important tools for the development of gene-drive systems to modify mosquito vector populations to control the transmission of pathogens that cause diseases such as malaria. However, one of the challenges for current Cas9-based drive systems is their ability to produce drive-resistant alleles resulting from insertions and deletions (indels) caused principally by nonhomologous end-joining following chromosome cleavage. Rapid increases in the frequency of such alleles may impair gene-drive dynamics. We explored the generation of indels in the germline and somatic cells in female gene-drive lineages using a series of selective crosses between a gene-drive line, AgNosCd-1, and wild-type mosquitoes. We find that potential drive-resistant mutant alleles are generated largely during embryonic development, most likely caused by deposition of the Cas9 endonuclease and guide RNAs in oocytes and resulting embryos by homozygous and hemizygous gene-drive mothers.
Expanding the flexibility of genome editing approaches for population control of the malaria mosquito
22226N. Kranjc, Imperial College London-PhD, 2022-04-01 15:50:55.
Discovery and adaptation of CRISPR-Cas systems for genome editing have allowed us to gain an efficient and yet simple tool for genetic manipulation in various fields of molecular biology and biotechnology. One of the most promising applications is the use of CRISPR-Cas9 endonuclease for gene drive systems as a population control strategy for various insect pests of medical and agricultural importance. Use of CRISPR-Cas9 endonuclease in gene drive applications has shown great promise in the laboratory, particularly for the control of Anopheles gambiae, the major vector of malaria. However, the performance of such gene drives can be limited by the range of available target sequences and by a propensity of existing endonuclease formulations to generate resistant mutations that hinder the gene drive’s efficiency. To expand the flexibility of gene drive systems, computational analysis was performed to identify additional Cas9 orthologs and their specificities that could usefully augment the targeting range of endonuclease-based gene drives. Two alternative variants of CRISPR-Cas endonucleases found in the bacterial species Lactobacillus rhamnosus and Bacteroides fragilis were assessed for their potential to expand the targeting space in the genome Anopheles gambiae. In addition, a computational tool was developed that evaluates neighbouring sequences to the target site to measure both its likely functional constraint and its likely propensity for DNA repair that could generate in-frame alleles. Using this approach we were able to generate a prioritized list of Anopheles gambiae target sites for gene drive applications that are less likely to be compromised by resistant alleles.
Toward a CRISPR-Cas9-Based Gene Drive in the Diamondback Moth Plutella xylostella
24426X. Xu, T. Harvey-Samuel, H. A. Siddiqui, J. X. D. Ang, M. E. Anderson, C. M. Reitmayer, E. Lovett, P. T. Leftwich, M. You and L. Alphey, The CRISPR Journal, 5:224-236. 2022-04-01 10:25:39.
Promising to provide powerful genetic control tools, gene drives have been constructed in multiple dipteran insects, yeast, and mice for the purposes of population elimination or modification. However, it remains unclear whether these techniques can be applied to lepidopterans. Here, we used endogenous regulatory elements to drive Cas9 and single guide RNA (sgRNA) expression in the diamondback moth (DBM), Plutella xylostella, and test the first split gene drive system in a lepidopteran. The DBM is an economically important global agriculture pest of cruciferous crops and has developed severe resistance to various insecticides, making it a prime candidate for such novel control strategy development. A very high level of somatic editing was observed in Cas9/sgRNA transheterozygotes, although no significant homing was revealed in the subsequent generation. Although heritable Cas9-medated germline cleavage as well as maternal and paternal Cas9 deposition were observed, rates were far lower than for somatic cleavage events, indicating robust somatic but limited germline activity of Cas9/sgRNA under the control of selected regulatory elements. Our results provide valuable experience, paving the way for future construction of gene drives or other Cas9-based genetic control strategies in DBM and other lepidopterans.
CRISPR-mediated knockout of cardinal and cinnabar eye pigmentation genes in the western tarnished plant bug
21243C. C. Heu, R. J. Gross, K. P. Le, D. M. LeRoy, B. Fan, J. J. Hull, C. S. Brent and J. A. Fabrick, Scientific Reports, 12. 2022-03-22 06:19:14.
The western tarnished plant bug, Lygus hesperus, is a key hemipteran pest of numerous agricultural, horticultural, and industrial crops in the western United States and Mexico. A lack of genetic tools in L. hesperus hinders progress in functional genomics and in developing innovative pest control methods such as gene drive. Here, using RNA interference (RNAi) against cardinal (LhCd), cinnabar (LhCn), and white (LhW), we showed that knockdown of LhW was lethal to developing embryos, while knockdown of LhCd or LhCn produced bright red eye phenotypes, in contrast to wild-type brown eyes. We further used CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated) genome editing to generate germline knockouts of both LhCd (Card) and LhCn (Cinn), producing separate strains of L. hesperus characterized by mutant eye phenotypes. Although the cardinal knockout strain Card exhibited a gradual darkening of the eyes to brown typical of the wild-type line later in nymphal development, we observed bright red eyes throughout all life stages in the cinnabar knockout strain Cinn, making it a viable marker for tracking gene editing in L. hesperus. These results provide evidence that CRISPR/Cas9 gene editing functions in L. hesperus and that eye pigmentation genes are useful for tracking the successful genetic manipulation of this insect.
Modelling homing suppression gene drive in haplodiploid organisms
21016Y. Liu and J. Champer, bioRxiv, 2021.10.12.464047. 2022-03-15 06:38:01.
Gene drives have shown great promise for suppression of pest populations. These engineered alleles can function by a variety of mechanisms, but the most common is the CRISPR homing drive, which converts wild-type alleles to drive alleles in the germline of heterozygotes. Some potential target species are haplodiploid, in which males develop from unfertilized eggs and thus have only one copy of each chromosome. This prevents drive conversion, a substantial disadvantage compared to diploids where drive conversion can take place in both sexes. Here, we study homing suppression gene drives in haplodiploids and find that a drive targeting a female fertility gene could still be successful. However, such drives are less powerful than in diploids and suffer more from functional resistance alleles. They are substantially more vulnerable to high resistance allele formation in the embryo due to maternally deposited Cas9 and gRNA and also to somatic cleavage activity. Examining spatial models where organisms move over a continuous landscape, we find that haplodiploid suppression drives surprisingly perform nearly as well as in diploids, possibly due to their ability to spread further before inducing strong suppression. Together, these results indicate that gene drive can potentially be used to effectively suppress haplodiploid populations.Competing Interest StatementThe authors have declared no competing interest.
Considerations for homology-based DNA repair in mosquitoes: Impact of sequence heterology and donor template source
20478J. X. D. Ang, K. Nevard, R. Ireland, D.-K. Purusothaman, S. A. N. Verkuijl, L. Shackleford, E. Gonzalez, M. A. E. Anderson and L. Alphey, PLOS Genetics, 18:e1010060. 2022-02-18 08:29:29.
Author summary The field of genetic control of mosquito vectors has progressed rapidly in recent years, especially in Cas9-based control systems, due to its robustness to elicit a species-specific and dispersive control of mosquito population. To generate a Cas9-based integration, Cas9 and sgRNA are used to cleave a chromosomal locus while a plasmid DNA donor, containing a genetic cargo flanked by sequences homologous to the chromosomal locus, is supplied as a repair template. This results in the cargo being copied into the genome through HDR. This form of integration, however, is currently one of the major bottlenecks for researchers as it involves a laborious process of microinjecting mosquito embryos and has rather low integration rates. In this study, we assessed the effects of homologous sequence mismatches and various donor template forms (i.e. plasmid, ssDNA, biotinylated ds/ssDNA) on HDR. We found that sequence mismatches and non-plasmid donors reduced the efficiency and integrity of integration, respectively. By analysing the direction and length of homologous sequence that was copied into the genome concurrently with the cargo, we inferred the mechanism responsible for the integrations observed in our study. These findings will be useful to guide future construct designs for optimal HDR rates in mosquitoes.
Gene drives for vertebrate pest control: realistic spatial modelling of eradication probabilities and times for island mouse populations
20171A. Birand, P. Cassey, J. V. Ross, J. C. Russell, P. Thomas and T. A. A. Prowse, Molecular Ecology, 2022-01-24 09:06:03.
Abstract Invasive alien species continue to threaten global biodiversity. CRISPR-based gene drives, which can theoretically spread through populations despite imparting a fitness cost, could be used to suppress or eradicate pest populations. We develop an individual-based, spatially explicit, stochastic model to simulate the ability of CRISPR-based homing and X-chromosome shredding drives to eradicate populations of invasive house mice (Mus muculus) from islands. Using the model, we explore the interactive effect of the efficiency of the drive constructs and the spatial ecology of the target population on the outcome of a gene-drive release. We also consider the impact of polyandrous mating and sperm competition, which could compromise the efficacy of some gene-drive strategies. Our results show that both drive strategies could be used to eradicate large populations of mice. Whereas parameters related to drive efficiency and demography strongly influence drive performance, we find that sperm competition following polyandrous mating is unlikely to impact the outcome of an eradication effort substantially. Assumptions regarding the spatial ecology of mice influenced the probability of and time required for eradication, with short-range dispersal capacities and limited mate-search areas producing `chase' dynamics across the island characterised by cycles of local extinction and recolonization by mice. We also show that highly efficient drives are not always optimal, when dispersal and mate-search capabilities are low. Rapid local population suppression around the introduction sites can cause loss of the gene drive before it can spread to the entire island. We conclude that, although the design of efficient gene drives is undoubtedly critical, accurate data on the spatial ecology of target species is critical for predicting the result of a gene-drive release.
Could we delete diseases passed down through our DNA?
20278E. Rayne, SYFY, 2022-01-23 11:33:23.
What has now been proven possible was once the stuff of science fiction dreams. CRISPR has shown it can successfully edit out detrimental genetic conditions before they are inherited — which could mean the beginning of the end for hereditary diseases. It could also help obliterate invasive species from ecosystems under attack. Imagine if gene editing could delete conditions you never asked to be born with while getting rid of the cane toad invasion in Australia. CRISPR-Cas9 has been able to successfully edit DNA again and again, but it’s never done anything like this. Kind of like DNA autocorrecting itself, the editing needs to make deletion in a cell happen during a certain phase of meiosis. Researcher Kimberly Cooper of UC San Diego, who coauthored a study recently published in PLOS Biology, figured out exactly when to get to that meiotic window and how to control which copies of genes are handed down to the next generation.
Analysis of a Cas12a-based gene-drive system in budding yeast
20029I. C. Lewis, Y. Yan and G. C. Finnigan, Access Microbiol, 3:000301. 2022-01-14 09:19:55.
The discovery and adaptation of CRISPR/Cas systems within molecular biology has provided advances across biological research, agriculture and human health. Genomic manipulation through use of a CRISPR nuclease and programmed guide RNAs has become a common and widely accessible practice. The identification and introduction of new engineered variants and orthologues of Cas9 as well as alternative CRISPR systems such as the type V group have provided additional molecular options for editing. These include distinct PAM requirements, staggered DNA double-strand break formation, and the ability to multiplex guide RNAs from a single expression construct. Use of CRISPR/Cas has allowed for the construction and testing of a powerful genetic architecture known as a gene drive within eukaryotic model systems. Our previous work developed a drive within budding yeast using Streptococcus pyogenes Cas9. Here, we installed the type V Francisella novicida Cas12a (Cpf1) nuclease gene and its corresponding guide RNA to power a highly efficient artificial gene drive in diploid yeast. We examined the consequence of altering guide length or introduction of individual mutational substitutions to the crRNA sequence. Cas12a-dependent gene-drive function required a guide RNA of at least 18 bp and could not tolerate most changes within the 5' end of the crRNA.
Scientists expand CRISPR-Cas9 genetic inheritance control in mammals
20038M. Aguilera, Phys Org, 2022-01-12 09:43:07.
Led by graduate student Alexander Weitzel, Grunwald, Cooper and their colleagues have now succeeded in developing CRISPR-Cas9 inheritance control in male mice by shifting the gene editing window to more closely match the timing of meiosis in both sexes. Their results were published December 23, 2021 in the journal PLOS Biology. The achievement advances the prospects of scientists being able to use genetic editing for new laboratory models in an array of research pursuits, from investigations of human disease to therapeutic drug design to invasive species removal. "For these gene conversion strategies to work in any context—in the lab or in wild populations—you need the mechanism of gene conversion to work in both males and females," said Cooper, associate professor in the Section of Cell and Developmental Biology, Division of Biological Sciences. "It seems as though the reason this process was previously working in females is because we were closer to the female meiotic window. Now that we've moved Cas9 expression to within the meiotic window in males, it works in them too."
Insect Allies – Assessment of a Viral Approach to Plant Genome Editing
20036K. Pfeifer, J. L. Frieß and B. Giese, Integrated Environmental Assessment and Management, 2022-01-12 09:38:06.
The DARPA program Insect Allies has already sparked scientific debate concerning technology assessment-related issues, among which the most prevalent is that of dual use potential. As apart from the issues concerning peaceful applications, the technology also provides the blueprint for a potential bioweapon as further evidenced by a recent publication. However, the combination of a virus-induced genetic modification of crop plants in the field using genetically modified insect vectors poses an increased risk potential in comparison to the hitherto existing use of genetically modified organisms. The technology's high depth of intervention enables a number of sources for hazard and a by trend high exposure, but it is also encumbered with notable deficits in knowledge. These issues call for a thorough technology assessment. This article aims to provide an initial characterization from a technology assessment perspective, focusing on potential sources of risk for this novel invasive environmental biotechnology at an early stage of research and development. This article is protected by copyright. All rights reserved.© 2022 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).
Reversing insecticide resistance with allelic-drive in Drosophila melanogaster
20085B. Kaduskar, R. B. S. Kushwah, A. Auradkar, A. Guichard, M. Li, J. B. Bennett, A. H. F. Julio, J. M. Marshall, C. Montell and E. Bier, Nature Communications, 13:291. 2022-01-12 09:16:33.
A recurring target-site mutation identified in various pests and disease vectors alters the voltage gated sodium channel (vgsc) gene (often referred to as knockdown resistance or kdr) to confer resistance to commonly used insecticides, pyrethroids and DDT. The ubiquity of kdr mutations poses a major global threat to the continued use of insecticides as a means for vector control. In this study, we generate common kdr mutations in isogenic laboratory Drosophila strains using CRISPR/Cas9 editing. We identify differential sensitivities to permethrin and DDT versus deltamethrin among these mutants as well as contrasting physiological consequences of two different kdr mutations. Importantly, we apply a CRISPR-based allelic-drive to replace a resistant kdr mutation with a susceptible wild-type counterpart in population cages. This successful proof-of-principle opens-up numerous possibilities including targeted reversion of insecticide-resistant populations to a native susceptible state or replacement of malaria transmitting mosquitoes with those bearing naturally occurring parasite resistant alleles.
Meiotic Cas9 expression mediates gene conversion in the male and female mouse germline
19726A. J. Weitzel, H. A. Grunwald, C. Weber, R. Levina, V. M. Gantz, S. M. Hedrick, E. Bier and K. L. Cooper, PLOS Biology, 19:e3001478. 2021-12-23 12:46:23.
Highly efficient gene conversion systems have the potential to facilitate the study of complex genetic traits using laboratory mice and, if implemented as a “gene drive,” to limit loss of biodiversity and disease transmission caused by wild rodent populations. We previously showed that such a system of gene conversion from heterozygous to homozygous after a sequence targeted CRISPR/Cas9 double-strand DNA break (DSB) is feasible in the female mouse germline. In the male germline, however, all DSBs were instead repaired by end joining (EJ) mechanisms to form an “insertion/deletion” (indel) mutation. These observations suggested that timing Cas9 expression to coincide with meiosis I is critical to favor conditions when homologous chromosomes are aligned and interchromosomal homologydirected repair (HDR) mechanisms predominate. Here, using a Cas9 knock-in allele at the Spo11 locus, we show that meiotic expression of Cas9 does indeed mediate gene conversion in the male as well as in the female germline. However, the low frequency of both HDR and indel mutation in both male and female germlines suggests that Cas9 may be expressed from the Spo11 locus at levels too low for efficient DSB formation. We suggest that more robust Cas9 expression initiated during early meiosis I may improve the efficiency of gene conversion and further increase the rate of “super-mendelian” inheritance from both male and female mice.
Scientists Used CRISPR Gene Editing to Choose the Sex of Mouse Pups
19906S. Fan, Singuarity Hub, 2021-12-23 08:37:01.
“Do you want a boy or a girl?” can be an awkward question.But in certain circles, it’s a question that’s asked every day. Take agriculture. In a perfect world, most cows would only birth females. Chicks would grow up to be all hens. “Sexing” a farm animal when they’re at a young age wouldn’t be a thing—especially when it means male animals, without the ability to produce milk or eggs, are often culled at a young age to preserve resources. There might be a better way. This month, a team tapped into the power of CRISPR to control the sex of the offspring in mice. By splicing CRISPR components into the parents’ genome, the team was able to flip on—or off—a switch that nearly perfectly determined the sex of their litters. Unlike previous attempts, the baby mice could go on to have litters of their own of both sexes. The targeted gene used for the edit is conserved across evolution, suggesting the technique could work in more animals than just mice. But it’s controversial. Essentially, the technique selectively kills off embryos of a certain sex, which immediately raises ethical red flags. For now, scientists aren’t concerned about the technology being used in humans due to its complexity. But the study is the latest to showcase biotech’s increasing ability to manipulate reproduction.
Exploiting a Y chromosome-linked Cas9 for sex selection and gene drive
19623S. Gamez, D. Chaverra-Rodriguez, A. Buchman, N. P. Kandul, S. C. Mendez-Sanchez, J. B. Bennett, C. H. Sánchez, T. Yang, I. Antoshechkin, J. E. Duque, P. A. Papathanos, J. M. Marshall and O. S. Akbari, Nature Communications, 7202. 2021-12-10 21:34:38.
CRISPR-based genetic engineering tools aimed to bias sex ratios, or drive effector genes into animal populations, often integrate the transgenes into autosomal chromosomes. However, in species with heterogametic sex chromsomes (e.g. XY, ZW), sex linkage of endonucleases could be beneficial to drive the expression in a sex-specific manner to produce genetic sexing systems, sex ratio distorters, or even sex-specific gene drives, for example. To explore this possibility, here we develop a transgenic line of Drosophila melanogaster expressing Cas9 from the Y chromosome. We functionally characterize the utility of this strain for both sex selection and gene drive finding it to be quite effective. To explore its utility for population control, we built mathematical models illustrating its dynamics as compared to other state-of-the-art systems designed for both population modification and suppression. Taken together, our results contribute to the development of current CRISPR genetic control tools and demonstrate the utility of using sex-linked Cas9 strains for genetic control of animals.
Genomic insertion locus and Cas9 expression in the germline affect CRISPR/Cas9-based gene drive performance in the yellow fever mosquito Aedes aegypti
19603W. R. Reid, J. Lin, A. E. Williams, R. Juncu, K. E. Olson and A. W. E. Franz, bioRxiv, 2021.12.08.471839. 2021-12-08 18:56:04.
The yellow fever mosquito Aedes aegypti is a major vector of arthropod-borne viruses, including dengue, chikungunya, and Zika. A novel approach to mitigate arboviral infections is to generate mosquitoes refractory to infection by overexpressing antiviral effector molecules. Such an approach requires a mechanism to spread these antiviral effectors through a population, for example, by using CRISPR/Cas9-based gene drive systems. Here we report an autonomous single-component gene drive system in Ae. aegypti that is designed for persistent population replacement. Critical to the design of a single-locus autonomous gene drive is that the selected genomic locus be amenable to both gene drive and the appropriate expression of the antiviral effector. In our study, we took a reverse engineering approach to target two genomic loci ideal for the expression of antiviral effectors and further investigated the use of three promoters for Cas9 expression (nanos, β2-tubulin, or zpg) for the gene drive. We found that both promoter selection and genomic target site strongly influenced the efficiency of the drive, resulting in 100% inheritance in some crosses. We also observed the formation of inheritable gene drive blocking indels (GDBI) in the genomic locus with the highest levels of gene drive. Overall, our drive system forms a platform for the further testing of driving antipathogen effector genes through Ae. aegypti populations.Competing Interest StatementThe authors have declared no competing interest.
Gene editing used to create all-male or all-female litters of mice
19542J. Goodyer, Science Focus, 2021-12-06 20:26:39.
As males are unable to produce milk or lay eggs, the ability to breed cows and hens that produce all-female litters is likely to be high on most poultry and dairy farmers’ wish lists. Now, scientists at the Francis Crick Institute and the University of Kent have come a step closer to realising this goal after successfully using CRISPR gene editing techniques to produce all-female or all-male litters of mice. The technique could also be used to improve animal welfare in areas of scientific research in which only male or only female animals are required for studies, the researchers say. To make the breakthrough the researchers took advantage of the fact that CRISPR consists of two parts – the Cas9 enzyme, which cuts the DNA and enables scientists to alter specific regions of genes, and the guide RNA, which carries the Cas9 enzyme to the desired region on the genome.
Genetic conversion of a split-drive into a full-drive element
19540G. Terradas, J. B. Bennett, Z. Li, J. M. Marshall and E. Bier, bioRxiv, 2021.12.05.471291. 2021-12-06 20:21:27.
Gene-drive systems offer an important new avenue for spreading beneficial traits into wild populations. Their core components, Cas9 and guide RNA (gRNA), can either be linked within a single cassette (full gene drive, fGD) or provided in two separate elements (split gene drive, sGD) wherein the gRNA-bearing element drives in the presence of an independent static source of Cas9. We previously designed a system engineered to turn split into full gene drives. Here, we provide experimental proof-of-principle for such a convertible system inserted at the spo11 locus, which is recoded to restore gene function. In multigenerational cage studies, the reconstituted spo11 fGD cassette initially drives with slower kinetics than the unlinked sGD element (using the same Mendelian vasa-Cas9 source), but eventually reaches a similar level of final introgression. Different kinetic behaviors may result from transient fitness costs associated with individuals co-inheriting Cas9 and gRNA transgenes during the drive process.
Gene editing used to create all-male or all-female mice litters
19502A. Reis, European Scientist, 2021-12-04 17:04:50.
Researchers from the Francis Crick Institute and the University of Kent used gene-editing technologies to create male-only and female-only mice litters, according to a study published in Nature Communications (1). The authors also suggested ways in which this method could be used to improve animal welfare in scientific research and agriculture. There are many situations in research and agriculture where it would be desirable to have just females or just males. For example, reproductive studies require only animals of the gender being studied, while in farming, egg and milk production needs only female animals. Sadly, in many cases, the unwanted animals end up being culled. “This work could have an immediate and valuable impact in scientific laboratories, as we’ve shown how it is safe and effective in mice, a common mammal used in medical and scientific research. While a lot of research needs both sexes, there are areas of study where only one is needed. For example, when studying the reproductive system, sex-specific diseases, or certain hormones”, said James Turner, group leader of the Sex Chromosome Biology Laboratory at the Crick says:
Gene-editing used to create single sex mice litters
19504The Francis Crick Institute, Phys Org, 2021-12-03 17:09:04.
Scientists at the Francis Crick Institute, in collaboration with University of Kent, have used gene editing technology to create female-only and male-only mice litters with 100% efficiency. This proof of principle study, published in Nature Communications today, demonstrates how the technology could be used to improve animal welfare in scientific research and perhaps also agriculture. In scientific research and also farming, there is often a need for either male or female animals. For example, laboratory research into male or female reproduction requires only animals of the sex being studied. And in farming, only female animals are required for egg production and in dairy herds. This means it is common practice for animals of the unrequired sex to be culled after birth. The researchers' new method uses a two-part genetic system to inactivate embryos shortly after fertilisation, allowing only the desired sex to develop. Such a genetically-based method to control the sex of offspring could drastically reduce culling in both industries. The embryo selection is based on the fact that there are two elements of CRISPR-Cas9—the Cas9 enzyme that cuts the DNA, allowing scientists to alter specific regions, and the guide RNA which carries the Cas9 to the right location on the genome. The team placed one element of the system on the father's X or Y chromosome, meaning that it will only be inherited by female or male embryos respectively. The other element is contributed by the mother, and is inherited by all embryos.
Single-sex mice litters were created with 100% efficiency using gene editing.
19498R. Silman, Brinkwire, 2021-12-03 17:00:12.
The Francis Crick Institute, in partnership with the University of Kent, has employed gene editing technology to construct 100% efficient female-only and male-only mouse litters. This proof-of-concept study, which was published today (Friday, December 3rd, 2021) in Nature Communications, shows how the technique could be used to improve animal wellbeing in scientific research and possibly agriculture. Male and female animals are frequently required in scientific research and husbandry. Laboratory research into male or female reproduction, for example, necessitates only animals of the examined sex. In addition, only female animals are needed for egg production and dairy herds in farming. This means that animals of the unrequired sex are routinely culled after birth.
Lab animals: Gene-editing technology is used to create female-only and male-only mice litters
19493todayuknews, Today UK News, 2021-12-03 16:50:32.
Single-sex litters of mice — comprising only either female or male pups — have been produced by means of so-called CRISPR-Cas9 gene editing technology. The technique, developed by experts at the Francis Crick Institute and the University of Kent, works by inactivating embryos of one sex shortly after fertilisation. It could be used to improve animal welfare in both laboratory and agricultural settings where, for various reasons, only female or male animals are needed. It is common for animals of the unrequired sex to be culled — a practice which could be drastically reduced by controlling the sex of the animals prior to birth. As the technique requires the genetic modification of both parents to work, however, the approach would not be suitable for forcing the sex of designer babies. Single-sex litters of mice — comprising only either female or male pups — have been produced by means of so-called CRISPR-Cas9 gene editing technology. Pictured: the mice that were bred to create single-sex litters. The black parts of their coat are caused by the genetically modified cells, while the white parts come from the non-modified parts of their genome
Gene editing produces all-male or all-female litters of mice
19491E. Pennisi, Science, 2021-12-03 16:41:41.
In some farmers’ ideal world, cows would birth only females, sows would bear no boars, and chicks would all grow up to be hens. Such sex ratios would stop them from killing millions of male animals, which don’t produce eggs or milk. Now, scientists are a step closer to this reality. Researchers have harnessed the gene editor CRISPR to produce litters of mice all of one sex. That’s a potential boon to agriculture and may offer a more immediate advantage in scientific research. “The paper shows a state-of-the-art solution to producing single-sex species,” with “impressive results,” says Ehud Qimron, a CRISPR expert at Tel Aviv University who was not involved with the work. The impact for lab animals may be huge. “In the past 5 years around 25,000 papers were published using mice in sex-specific research studies,” says study co-author James Turner, a molecular geneticist at the Francis Crick Institute. “If we could prevent the generation of the unstudied sex, the number [saved] would be in the hundreds of thousands.” Other methods exist to skew the male/female ratio of newborn animals. Scientists can sort sperm by the weight of the sex chromosome, or cause embryos of one sex to die before birth. In a study published 2 years ago, researchers using the gene editor CRISPR managed to produce altered mice in which four of five litters were all female.
CRISPR-Cas9 effectors facilitate generation of single-sex litters and sex-specific phenotypes
19469C. Douglas, V. Maciulyte, J. Zohren, D. M. Snell, S. K. Mahadevaiah, O. A. Ojarikre, P. J. I. Ellis and J. M. A. Turner, Nature Communications, 12:6926. 2021-12-03 15:06:30.
Animals are essential genetic tools in scientific research and global resources in agriculture. In both arenas, a single sex is often required in surplus. The ethical and financial burden of producing and culling animals of the undesired sex is considerable. Using the mouse as a model, we develop a synthetic lethal, bicomponent CRISPR-Cas9 strategy that produces male- or female-only litters with one hundred percent efficiency. Strikingly, we observe a degree of litter size compensation relative to control matings, indicating that our system has the potential to increase the yield of the desired sex in comparison to standard breeding designs. The bicomponent system can also be repurposed to generate postnatal sex-specific phenotypes. Our approach, harnessing the technological applications of CRISPR-Cas9, may be applicable to other vertebrate species, and provides strides towards ethical improvements for laboratory research and agriculture.
CRISPR gene-drive systems based on Cas9 nickases promote super-Mendelian inheritance in Drosophila
19471V. Lopez del Amo, S. Sanz Juste and V. M. Gantz, bioRxiv, 2021.12.01.470847. 2021-12-02 15:07:14.
CRISPR-based gene drive systems can be used to modify entire wild populations due to their ability to bias their own inheritance towards super-Mendelian rates (>100%). Current gene drives contain a Cas9 and a gRNA gene inserted at the location targeted by the gRNA. These gene products are able to cut the opposing wildtype allele, and lead to its replacement with a copy of the gene drive through the homology-directed DNA repair pathway. When this allelic conversion occurs in the germline it leads to the preferential inheritance of the engineered allele; a property that has been proposed to disseminate engineered traits for managing disease-transmitting mosquito populations. Here, we report a novel gene-drive strategy relying on Cas9 nickases which operates by generating staggered paired-nicks in the DNA to promote propagation of the gene drive allele. We show that only when 5' overhangs are generated, the system efficiently leads to allelic conversion. Further, the nickase gene-drive arrangement produces large stereotyped deletions, providing potential advantages for targeting essential genes. Indeed, the nickase-gene-drive design should expand the options available for gene drive designs aimed at applications in mosquitoes and beyond.
Applying functional genomics to the study of lamprey development and sea lamprey population control
19946J. R. York, R. E. Thresher and D. W. McCauley, Journal of Great Lakes Research, 47:S639-S649. 2021-12-01 09:30:24.
Lampreys are one of the few survivors of an ancient lineage of jawless vertebrates and have become an important study organism in numerous disciplines in the biological sciences, including evolutionary biology, embryology, ecology, physiology and biomedicine. At the same time, however, lampreys have created economic and ecological problems due, primarily, to the invasion of parasitic sea lamprey (Petromyzon marinus) into the North American Great Lakes and consequent negative impacts on local fish populations. Barriers, trapping and lampricide treatments have reduced these impacts, but concern for habitat restoration, non-target effects and possible evolution of resistance to lampricides suggests the need to develop additional strategies that supplement current control measures. The advent of functional genomics, and in particular CRISPR/Cas9 genome editing, offers a molecular approach to this on-going problem. Here, we review the successful application of functional genetic, transcriptomic, and CRISPR/Cas9 genome editing technologies in lampreys to address basic research questions in the fields of evolutionary and developmental biology. We then describe how these tools may be repurposed for use by fishery and conservation biologists to approach the problem of invasive sea lamprey from a molecular-genetic perspective.
Genome editing and its applications for insect pest control: Curse or blessing?
19401Hacker, I. , and Schetelig, M. F, AREA-WIDE INTEGRATED PEST MANAGEMENT: Development and Field Application, 2021-11-29 18:00:39.
Gene and genome editing are described as cutting-edge research tools with the potential to tackle urgent global challenges in the management of agricultural pests and human disease vectors such as mosquitoes. The field is defined by the chances and challenges to interlink the disciplines of insect genomics, molecular biology, and pest control together with the need for clear risk assessment, policy development and public approval of the application of such novel technologies. The goal is to generate innovative and sustainable pest control solutions applied in the best interest for the environment and human society. Here, starting from available genome editing technologies, the current strategies and applications for insect pest control are discussed, including approaches to overcome the evolution of resistance alleles and other potential pitfalls to be expected from selective pressures resulting from gene drive applications. They are supplemented by views on regulatory, policy and ethical considerations that in our opinion will be necessary to define how the different tools can be used in the future in a safe and responsible way.
High-resolution in situ analysis of Cas9 germline transcript distributions in gene-drive Anopheles mosquitoes
19324G. Terradas, A. Hermann, A. A. James, W. McGinnis and E. Bier, G3-Genes Genomes Genetics, 2021-11-15 14:20:36.
Gene drives are programmable genetic elements that can spread beneficial traits into wild populations to aid in vector-borne pathogen control. Two different drives have been developed for population modification of mosquito vectors. The Reckh drive (vasa-Cas9) in Anopheles stephensi displays efficient allelic conversion through males but generates frequent drive-resistant mutant alleles when passed through females. In contrast, the AgNosCd-1 drive (nos-Cas9) in Anopheles gambiae achieves almost complete allelic conversion through both genders. Here, we examined the subcellular localization of RNA transcripts in the mosquito germline. In both transgenic lines, Cas9 is strictly coexpressed with endogenous genes in stem and premeiotic cells of the testes, where both drives display highly efficient conversion. However, we observed distinct colocalization patterns for the two drives in female reproductive tissues. These studies suggest potential determinants underlying efficient drive through the female germline. We also evaluated expression patterns of alternative germline genes for future gene-drive designs.
Temperature-Inducible Precision-Guided Sterile Insect Technique
19268N. P. Kandul, J. R. Liu and O. S. Akbari, CRISPR Journal, 14. 2021-11-03 16:11:04.
Releases of sterile males are the gold standard for many insect population control programs, and precise sex sorting to remove females prior to male releases is essential to the success of these operations. To advance traditional methods for scaling the generation of sterile males, we previously described a CRISPR-mediated precision-guided sterile insect technique (pgSIT), in which Cas9 and gRNA strains are genetically crossed to generate sterile males for mass release. While effective at generating F-1 sterile males, pgSIT requires a genetic cross between the two parental strains, which requires maintenance and sexing of two strains in a factory. Therefore, to advance pgSIT further by removing this crossing step, here we describe a next-generation temperature-inducible pgSIT (TI-pgSIT) technology and demonstrate its proof-of-concept in Drosophila melanogaster. Importantly, we were able to develop a true breeding strain for TI-pgSIT that eliminates the requirement for sex sorting-a feature that may help further automate production at scale.
Modeling the efficacy of CRISPR gene drive for schistosomiasis control
19109R. E. Grewelle, J. Perez-Saez, J. Tycko, E. K. O. Namigai, C. G. Rickards and G. A. De Leo, bioRxiv, 2021.10.29.466423. 2021-11-01 14:51:40.
CRISPR gene drives could revolutionize the control of infectious diseases by accelerating the spread of engineered traits that limit parasite transmission in wild populations. While much effort has been spent developing gene drives in mosquitoes, gene drive technology in molluscs has received little attention despite the role of freshwater snails as obligate, intermediate hosts of parasitic flukes causing schistosomiasis -- a disease of poverty affecting more than 200 million people worldwide. A successful drive in snails must overcome self-fertilization, which prevents a drive's spread. Simultaneous hermaphroditism is a feature of snails -- distinct from gene drive model organisms -- and is not yet incorporated in gene drive models of disease control. Here we developed a novel population genetic model accounting for snails' sexual and asexual reproduction, susceptibility to parasite infection regulated by multiple alleles, fitness differences between genotypes, and a range of drive characteristics. We then integrated this model with an epidemiological model of schistosomiasis transmission and snail population dynamics. Simulations showed that gene drive establishment can be hindered by a variety of biological and ecological factors, including selfing. However, our model suggests that, under a range of conditions, gene drive mediated immunity in snails could maintain rapid disease reduction achieved by annual chemotherapy treatment of the human population, leading to long-term elimination. These results indicate that gene drives, in coordination with existing public health measures, may become a useful tool to reduce schistosomiasis burden in selected transmission settings with effective CRISPR construct design and close evaluation of the genetic and ecological landscape.Competing Interest StatementJT and EKON were seed funded by the Merck Innovation Cup 2016 for research on schistosomiasis, and previously employed as external consultants to the Global Health Institute of Merck (KGaA) which produces treatments for schistosomiasis. REG and GADL were partially supported by the National Science Foundation's grants DEB-2011179 and ICER-2024383.
Genome Editing Tools and Gene Drives: A Brief Overview (1st ed.).
19295R. Mudziwapasi, R. Chekera, C. Z. Ncube, I. Shoko, B. Ncube, T. Moyo, J. G. Chimbo, J. Dube, F. F. Mashiri, M. A. Mubani, D. Maruta, C. Chimbo, M. Masuku, R. Shoko, R. P. Nyamusamba and F. N. Jomane, CRC Press, 2021-10-21 14:20:34.
Genome-editing methods are becoming routine tools for molecular and cell biologists. Such tools include ZFNs, CRISPR, megaTALs and TALENs. These tools are revolutionizing the creation of precisely manipulated genomes to modify the characteristics of organisms or cells. Additionally, gene drives have altered the way we understand inheritance laws. They give us the ability to have total control of the inheritance of traits of choice and importance. This succinct volume summarizes the history, principles and applications – as well as the advantages and disadvantages – of each of these tools and various kinds of gene drives. The book is part of a program to produce books helpful to students and faculties of science at colleges and universities. This volume in the Pocket Guides to Biomedical Sciences series will help demystify these technologies. The book fills the gap between established conventional methods and the novel and exciting newly introduced tools of genome editing and gene drives. It will help young scientists understand the emerging genome-editing tools and gene drives, thereby promoting related research and adoption.
Modeling homing suppression gene drive in haplodiploid organisms
18986Y. Liu and J. Champer, bioRxiv, 2021.10.12.464047. 2021-10-13 14:35:58.
Gene drives have shown great promise for suppression of pest populations. These engineered alleles can function by a variety of mechanisms, but the most common is the CRISPR homing drive, which converts wild-type alleles to drive alleles in the germline of heterozygotes. Some potential target species are haplodiploid, in which males develop from unfertilized eggs and thus have only one copy of each chromosome. This prevents drive conversion, a substantial disadvantage compared to diploids where drive conversion can take place in both sexes. Here, we study the characteristics of homing suppression gene drives in haplodiploids and find that a drive targeting a female fertility gene could still be successful. However, such drives are less powerful than in diploids. They are substantially more vulnerable to high resistance allele formation in the embryo due to maternally deposited Cas9 and gRNA and also to somatic cleavage activity. Examining models of continuous space where organisms move over a landscape, we find that haplodiploid suppression drives surprisingly perform nearly as well as in diploids, possibly due to their ability to spread further before inducing strong suppression. Together, these results indicate that gene drive can potentially be used to effectively suppress haplodiploid populations.
Gene drive: a faster route to plant improvement
18859H. A. Siddiqui, T. Harvey-Samuel and S. Mansoor, Trends in Plant Science, 2021-10-06 18:24:37.
Gene drives for control of vector-borne diseases have been demonstrated in insects but remain challenging in plants. Theoretically, they could be transformative in speeding breeding programs and contributing to food security through providing novel weed control methods. Zhang et al. now report the possibility of implementing gene drive in plants for the first time.
Towards CRISPR/Cas9-based gene drive in the diamondback moth Plutella xylostella
18867X. Xu, T. Harvey-Samuel, H. Siddiqui, J. Ang, M. A. E. Anderson, C. Reitmayer, E. Lovett, P. T. Leftwich, M. You and L. Alphey, bioRxiv, 2021.10.05.462963. 2021-10-05 19:03:11.
Promising to provide powerful genetic control tools, gene drives have been constructed in multiple dipterans, yeast and mice, for the purposes of population elimination or modification. However, it remains unclear whether these techniques can be applied to lepidopterans. Here, we used endogenous regulatory elements to drive Cas9 and sgRNA expression in the diamondback moth, (Plutella xylostella), and test the first split-drive system in a lepidopteran. The diamondback moth is an economically important global agriculture pest of cruciferous crops and has developed severe resistance to various insecticides, making it a prime candidate for such novel control strategy development. A very high level of somatic editing was observed in Cas9/sgRNA transheterozygotes, although no significant homing was revealed in the subsequent generation. Although heritable, Cas9-medated germline cleavage, as well as maternal and paternal Cas9 deposition was observed, rates were far lower than for somatic cleavage events, indicating robust somatic but limited germline activity of Cas9/sgRNA under the control of selected regulatory elements. Our results provide valuable experience, paving the way for future construction of gene drive-based genetic control strategies in DBM or other lepidopterans.Competing Interest StatementThe authors have declared no competing interest.
Scientists use gene editing tool to target mosquito-spread disease
18650Medical Research Council, Phys Org, 2021-09-13 20:12:56.
Advances in genome editing have allowed the development of genetic insect control methods, which could be highly effective and are species-specific. The results have been published in Scientific Reports. Scientists showed that a method involving a gene editing tool called CRISPR/Cas9 could be used to successfully introduce a gene for a fluorescent protein into the genome of southern house mosquitoes. The gene could be passed on to the next generation through mating. This is a vital component of generating genetic pest management tools. It will allow the desired traits (such as the inability to spread a disease or produce fertile offspring) to be spread throughout a population. The inserted gene produces red fluorescence proteins so that mosquitoes with one or more edited gene fluoresce red. Scientists targeted an eye color gene for the insertion site of the fluorescence gene so mosquitoes that inherited two edited genes from their parents would have white eyes, not black. Both these traits make it easier for scientists to easily identify mosquitoes whose genomes had been modified.
Mosquitoes Sterilized by CRISPR Powered Precision System
18648A. A. Sarkar, Genetic Engineering & Biotechnology News, 2021-09-13 20:06:53.
Each year millions around the world are infected by dengue, chikungunya, and Zika viruses. The principal culprit behind the transmission of these deadly diseases is the mosquito vector, Aedes aegypti. Conventional methods of pest control have so far fallen short. To curb the spread of A. aegypti, researchers at the University of California, San Diego (UCSD), have now developed a CRISPR-based molecular genetic control system called precision-guided sterile insect technique (pgSIT) that alters insect genes to generate flightless female and sterile male mosquitoes. The pgSIT system can be deployed effectively at any stage in the life cycle of the mosquito. The authors used mathematical models to empirically demonstrate that once released, male A. aegypti mosquitoes sterilized using the pgSIT system can compete, suppress, and eliminate fertile mosquito populations in the wild. The pgSIT system is not limited to restricting mosquito populations, it can be adapted to different vectors to curb transmissible diseases in a safe, confinable, and reversible manner, the authors claim. These findings are reported in the Nature Communications article, “Suppressing mosquito populations with precision guided sterile males.”
New precision-guided sterile insect technique designed to control disease-spreading mosquitoes
18674E. Henderson, News Medical Life Sciences, 2021-09-11 19:59:16.
Leveraging advancements in CRISPR-based genetic engineering, researchers at the University of California San Diego have created a new system that restrains populations of mosquitoes that infect millions each year with debilitating diseases. The new precision-guided sterile insect technique, or pgSIT, alters genes linked to male fertility--creating sterile offspring--and female flight in Aedes aegypti, the mosquito species responsible for spreading wide-ranging diseases including dengue fever, chikungunya and Zika.Details of the new pgSIT are described September 10, 2021, in the journal Nature Communications. pgSIT differs from "gene drive" systems that could suppress disease vectors by passing desired genetic alterations indefinitely from one generation to the next. Instead, pgSIT uses CRISPR to sterilize male mosquitoes and render female mosquitoes, which spread disease, as flightless. The system is self-limiting and is not predicted to persist or spread in the environment, two important safety features that should enable acceptance for this technology. Akbari says the envisioned pgSIT system could be implemented by deploying eggs of sterile males and flightless females at target locations where mosquito-borne disease spread is occurring.
Genetic engineering tech promises to sterilize disease-spreading mosquitoes
18525B. Hays, UPI, 2021-09-10 16:01:17.
Inspired by improvements in CRISPR-based genetic engineering, scientists have developed a more precise insect sterilization system to curtail, or even eliminate, disease-spreading Aedes aegypti mosquito populations. The so-called "precision-guided sterile insect technique," or pgSIT, relies on gene alterations that disrupt fertility in males and flight in females. Gene-altered males are released into a problematic population to compete with healthy males. "pgSIT is a new scalable genetic control system that uses a CRISPR-based approach to engineer deployable mosquitoes that can suppress populations," corresponding author Omar Akbari said in a press release. Map of malaria parasite's gene activity reveals new targets for drugs, vaccines "Males don't transmit diseases so the idea is that as you release more and more sterile males, you can suppress the population without relying on harmful chemicals and insecticides," said Akbari, a professor of biological sciences at the University of California, San Diego.
Genetic Engineering Technology Promises To Sterilize Disease-Spreading Mosquito Populations
18519D. Gyllhem, VIGOURTIMES, 2021-09-10 15:00:02.
Inspired by improvements in CRISPR-based genetic engineering, scientists have developed a more precise insect sterilization system to curtail, or even eliminate, disease-spreading Aedes aegypti mosquito populations. The so-called “precision-guided sterile insect technique,” or pgSIT, relies on gene alterations that disrupt fertility in males and flight in females. Gene-altered males are released into a problematic population to compete with healthy males. Scientists described the novel method in a new paper, published Friday in the journal Nature Communications. “pgSIT is a new scalable genetic control system that uses a CRISPR-based approach to engineer deployable mosquitoes that can suppress populations,” corresponding author Omar Akbari said in a press release. “Males don’t transmit diseases so the idea is that as you release more and more sterile males, you can suppress the population without relying on harmful chemicals and insecticides,” said Akbari, a professor of biological sciences at the University of California, San Diego.
New Technology Designed to Genetically Control Disease-spreading Mosquitoes
18515M. Aguilera, UC San Diego News Center, 2021-09-10 14:49:00.
Leveraging advancements in CRISPR-based genetic engineering, researchers at the University of California San Diego have created a new system that restrains populations of mosquitoes that infect millions each year with debilitating diseases. An illustration by study coauthor Stephanie Gamez depicts flightless females and sterile male mosquitoes, features of the new precision-guided sterile insect technique, or pgSIT, which is designed to control disease-spreading Aedes aegypti mosquitoes. The new precision-guided sterile insect technique, or pgSIT, alters genes linked to male fertility—creating sterile offspring—and female flight in Aedes aegypti, the mosquito species responsible for spreading wide-ranging diseases including dengue fever, chikungunya and Zika. “pgSIT is a new scalable genetic control system that uses a CRISPR-based approach to engineer deployable mosquitoes that can suppress populations,” said UC San Diego Biological Sciences Professor Omar Akbari. “Males don’t transmit diseases so the idea is that as you release more and more sterile males, you can suppress the population without relying on harmful chemicals and insecticides.”
Suppressing mosquito populations with precision guided sterile males
18513M. Li, T. Yang, M. Bui, S. Gamez, T. Wise, N. P. Kandul, J. Liu, L. Alcantara, H. Lee, J. R. Edula, R. Raban, Y. Zhan, Y. Wang, N. DeBeaubien, J. Chen, H. M. Sánchez C, J. B. Bennett, I. Antoshechkin, C. Montell, J. M. Marshall and O. S. Akbari, Nature Communications, 12:5374. 2021-09-10 14:42:45.
The mosquito Aedes aegypti is the principal vector for arboviruses including dengue/yellow fever, chikungunya, and Zika virus, infecting hundreds of millions of people annually. Unfortunately, traditional control methodologies are insufficient, so innovative control methods are needed. To complement existing measures, here we develop a molecular genetic control system termed precision-guided sterile insect technique (pgSIT) in Aedes aegypti. PgSIT uses a simple CRISPR-based approach to generate flightless females and sterile males that are deployable at any life stage. Supported by mathematical models, we empirically demonstrate that released pgSIT males can compete, suppress, and even eliminate mosquito populations. This platform technology could be used in the field, and adapted to many vectors, for controlling wild populations to curtail disease in a safe, confinable, and reversible manner.
Gene drive escape from resistance depends on mechanism and ecology
18261F. Cook, J. J. Bull and R. Gomulkiewicz, bioRxiv, 2021.08.30.458221. 2021-08-31 13:20:26.
Gene drives can potentially be used to suppress pest populations, and the advent of CRISPR technology has made it feasible to engineer them in many species, especially insects. What remains largely unknown for implementations is whether anti-drive resistance will evolve to block the population suppression. An especially serious threat to some kinds of drive is mutations in the CRISPR cleavage sequence that block the action of CRISPR, but designs have been proposed to avoid this type of resistance. Various types of resistance at loci away from the cleavage site remain a possibility, which is the focus here. It is known that modest-effect suppression drives can essentially `outrun' unlinked resistance even when that resistance is present from the start. We demonstrate here how the risk of evolving (unlinked) resistance can be further reduced without compromising overall suppression by introducing multiple suppression drives or by designing drives with specific ecological effects. However, we show that even modest-effect suppression drives remain vulnerable to the evolution of extreme levels of inbreeding, which halt the spread of the drive without actually interfering with its mechanism. The landscape of resistance evolution against suppression drives is therefore complex, but avenues exist for enhancing gene drive success.Competing Interest StatementThe authors have declared no competing interest.
Versatile Applications of the CRISPR/Cas Toolkit in Plant Pathology and Disease Management
19445M. S. Wheatley and Y. N. Yang, Phytopathology, 111:1080-1090. 2021-08-25 21:11:15.
New tools and advanced technologies have played key roles in facilitating basic research in plant pathology and practical approaches for disease management and crop health. Recently. the CRISPR/Cas (clustered regularly interspersed short palindromic repeats/CRISPR-associated) system has emerged as a powerful and versatile tool for genome editing and other molecular applications. This review aims to introduce and highlight the CRISPR/Cas toolkit and its current and future impact on plant pathology and disease management. We will cover the rapidly expanding horizon of various CRISPR/Cas applications in the basic study of plant-pathogen interactions, genome engineering of plant disease resistance, and molecular diagnosis of diverse pathogens. Using the citrus greening disease as an example, various CRISPR/Cas-enabled strategies are presented to precisely edit the host genome for disease resistance, to rapidly detect the pathogen for disease management, and to potentially use gene drive for insect population control. At the cutting edge of nucleic acid manipulation and detection, the CRISPR/Cas toolkit will accelerate plant breeding and reshape crop production and disease management as we face the challenges of 21st century agriculture.
Cas9-Mediated Gene-Editing in the Black-Legged Tick, Ixodes Scapularis, by Embryo Injection and ReMOT Control.
17197A. a. P. Sharma, Michael N. and Reyes, Jeremiah B. and Chana, Randeep and Yim, Won C. and Heu, Chan C. and Kim, Donghun and Chaverra-Rodriguez, Duverney and Rasgon, Jason L. and Harrell, Robert A. and Nuss, Andrew B. and Gulia-Nuss, Monika,, Cell Reports, 2021-08-20 20:35:08.
Despite their capacity to acquire and pass on an array of debilitating pathogens, research on ticks has lagged behind other arthropod vectors, such as mosquitoes, largely because of a lack of genetic and molecular tools. CRISPR-Cas9 is transforming non-model organism research; however, successful gene editing has not yet been reported in ticks. Technical challenges for injecting tick embryos to attempt gene editing have further slowed research progress. Herein we report a successful tick embryo injection protocol for the black-legged tick, Ixodes scapularis, the first for any chelicerate species, and the use of this protocol for genome editing with CRISPR-Cas9. We also demonstrated for the first time that the ReMOT Control technique can be successfully used to generate genome mutations outside Insecta. Our results provide innovative tools to the tick research community that are essential for advancing our understanding of the molecular mechanisms governing pathogen transmission by tick vectors as well as for understanding the underlying biology of host-vector-pathogen interactions.
CRISPR/Cas9-based functional characterization of the pigmentation gene ebony in Plutella xylostella
18155X. Xu, T. Harvey-Samuel, J. Yang, M. You and L. Alphey, Insect Molecular Biology, 2021-08-20 17:59:44.
Abstract Body pigmentation is an important character of insects in adapting to biotic and abiotic environmental challenges. Additionally, based on the relative ease of screening, several genes involved in insect melanisation have been used in classic genetic studies or as visual markers in constructing transgenic insects. Here, a homolog of the Bombyx mori melanisation-inhibiting gene ebony, associated with the conversion of dopamine to N-?-alanyl dopamine, was identified in a global pest, Plutella xylostella. The CRISPR/Cas9 system was applied to generate multiple Pxebony knockout alleles which were crossed to produce a Pxebony knockout strain, showing darker pigmentation in larvae, pupae and adults, compared with wildtype. Interestingly, we observed that Pxebony heterozygotes displayed an intermediate darkened phenotype, indicating partial dominance between the knockout and wildtype alleles. The fitness costs of Pxebony-deficiency were also assessed in the mutant strain, indicating that embryo hatchability and larval survival were significantly reduced, while the eclosion rate was not obviously affected. Our work provides a potential target for exploring CRISPR-based genetics-control systems in this economically important pest lepidopteran.
Gene drives gaining speed
17972E. Bier, Nature Reviews Genetics, 2021-08-06 13:50:56.
Gene drives are selfish genetic elements that are transmitted to progeny at super-Mendelian (>50%) frequencies. Recently developed CRISPR–Cas9-based gene-drive systems are highly efficient in laboratory settings, offering the potential to reduce the prevalence of vector-borne diseases, crop pests and non-native invasive species. However, concerns have been raised regarding the potential unintended impacts of gene-drive systems. This Review summarizes the phenomenal progress in this field, focusing on optimal design features for full-drive elements (drives with linked Cas9 and guide RNA components) that either suppress target mosquito populations or modify them to prevent pathogen transmission, allelic drives for updating genetic elements, mitigating strategies including trans-complementing split-drives and genetic neutralizing elements, and the adaptation of drive technology to other organisms. These scientific advances, combined with ethical and social considerations, will facilitate the transparent and responsible advancement of these technologies towards field implementation.
The Promise of Genetics and Genomics for Improving Invasive Mammal Management on Islands
17936B. T. Burgess, R. L. Irvine, G. R. Howald and M. A. Russello, Frontiers in Ecology and Evolution, 9. 2021-08-03 13:21:15.
Invasive species are major contributors to global biodiversity decline. Invasive mammalian species (IMS), in particular, have profound negative effects in island systems that contain disproportionally high levels of species richness and endemism. The eradication and control of IMS have become important conservation tools for managing species invasions on islands, yet these management operations are often subject to failure due to knowledge gaps surrounding species- and system-specific characteristics, including invasion pathways and contemporary migration patterns. Here, we synthesize the literature on ways in which genetic and genomic tools have effectively informed IMS management on islands, specifically associated with the development and modification of biosecurity protocols, and the design and implementation of eradication and control programs. In spite of their demonstrated utility, we then explore the challenges that are preventing genetics and genomics from being implemented more frequently in IMS management operations from both academic and non-academic perspectives, and suggest possible solutions for breaking down these barriers. Finally, we discuss the potential application of genome editing to the future management of invasive species on islands, including the current state of the field and why islands may be effective targets for this emerging technology.
Resistance to a CRISPR-based gene drive at an evolutionarily conserved site is revealed by mimicking genotype fixation
17940S. Fuchs, W. Garrood, A. Beber, A. Hammond, R. Galizi, M. Gribble, G. Morselli, T.-Y. Hui, K. Willis, N. Kranjc, A. Burt, T. Nolan and A. Crisanti, bioRxiv, 2021-07-26 13:48:52.
CRISPR-based homing gene drives can be designed to disrupt essential genes whilst biasing their own inheritance, leading to suppression of mosquito populations in the laboratory. This class of gene drives relies on CRISPR-Cas9 cleavage of a target sequence and copying (‘homing’) therein of the gene drive element from the homologous chromosome. However, target site mutations that are resistant to cleavage yet maintain the function of the essential gene are expected to be strongly selected for. Targeting functionally constrained regions where mutations are not easily tolerated should lower the probability of resistance. Evolutionary conservation at the sequence level is often a reliable indicator of functional constraint, though the actual level of underlying constraint between one conserved sequence and another can vary widely. Here we generated a novel gene drive in the malaria vector Anopheles gambiae , targeting an ultra-conserved target site in a haplosufficient essential gene (AGAP029113) required during mosquito development, which fulfils many of the criteria for the target of a population suppression gene drive. We then designed a selection regime to experimentally assess the likelihood of generation and subsequent selection of gene drive resistant mutations at its target site. We simulated, in a caged population, a scenario where the gene drive was approaching fixation, where selection for resistance is expected to be strongest. Continuous sampling of the target locus revealed that a single, restorative, in-frame nucleotide substitution was selected. Our findings show that ultra-conservation alone need not be predictive of a site that is refractory to target site resistance. Our strategy to evaluate resistance in vivo could help to validate candidate gene drive targets for their resilience to resistance and help to improve predictions of the invasion dynamics of gene drives in field populations. <h4>Author summary</h4> Gene drives have the potential to be applied as novel control strategy of disease-transmitting mosquitoes, by spreading genetic traits that suppress or modify the target population. Many gene drive elements work by recognising and cutting a specific target sequence in the mosquito genome and copying themselves into that target sequence allowing the gene drive to increase in frequency in the population. Like other mosquito control interventions, efficacy will greatly depend on minimising the development of resistance to the gene drive mechanism - most likely via a change in the target sequence that prevents further cutting. One strategy to reduce resistance is to target sequences that are highly conserved, which implies that changes cannot easily be tolerated. We developed a strategy that simulates high selection pressure, under which resistance is most likely to emerge, and therefore provides a stringent test of its propensity to arise. Unlike previous results with another gene drive, we recovered a resistant allele within a few generations of gene drive exposure and at high frequency. Our results show that conserved sequences can vary hugely in ability to tolerate mutations and highlights the need to functionally validate future candidate gene drive target sites for their robustness to resistance.
CRISPR/Cas-9 mediated knock-in by homology dependent repair in the West Nile Virus vector Culex quinquefasciatus Say
17811D.-K. Purusothaman, L. Shackleford, M. A. E. Anderson, T. Harvey-Samuel and L. Alphey, Scientific Reports, 11:14964. 2021-07-22 14:14:08.
Culex quinquefasciatus Say is a mosquito distributed in both tropical and subtropical regions of the world. It is a night-active, opportunistic blood-feeder and vectors many animal and human diseases, including West Nile Virus and avian malaria. Current vector control methods (e.g. physical/chemical) are increasingly ineffective; use of insecticides also imposes hazards to both human and ecosystem health. Advances in genome editing have allowed the development of genetic insect control methods, which are species-specific and, theoretically, highly effective. CRISPR/Cas9 is a bacteria-derived programmable gene editing tool that is functional in a range of species. We describe the first successful germline gene knock-in by homology dependent repair in C. quinquefasciatus. Using CRISPR/Cas9, we integrated an sgRNA expression cassette and marker gene encoding a fluorescent protein fluorophore (Hr5/IE1-DsRed, Cq7SK-sgRNA) into the kynurenine 3-monooxygenase (kmo) gene. We achieved a minimum transformation rate of 2.8%, similar to rates in other mosquito species. Precise knock-in at the intended locus was confirmed. Insertion homozygotes displayed a white eye phenotype in early-mid larvae and a recessive lethal phenotype by pupation. This work provides an efficient method for engineering C. quinquefasciatus, providing a new tool for developing genetic control tools for this vector.
A Sterile Solution: How Crispr Could Protect Wild Salmon
17821L. Abend, UNDARK, 2021-07-21 14:44:32.
In an attempt to prevent escaped fish from interbreeding with their wild counterparts and threatening the latter’s genetic diversity, molecular biologist Anna Wargelius and her team at the Institute of Marine Research in Norway have spent years working on ways to induce sterility in Atlantic salmon. Farmed salmon that cannot reproduce, after all, pose no threat to the gene pool of wild stocks, and Wargelius has successfully developed a technique that uses the gene-editing technology Crispr to prevent the development of the cells that would otherwise generate functioning sex organs. In fact, Wargelius’ team was a little too successful. To be financially viable, commercial fish farms need at least some of their stock to reproduce. So the scientists went a step further, developing a method of temporarily reversing the modification they had already made. They’ve created what they call “sterile parents.” The term may sound like an oxymoron, but the sterile parents have the potential to solve one of the most pressing problems facing salmon aquaculture, both in Norway and around the world. Wargelius says it could be up to a decade before the results of her work are commercially available, but once they are, they have the potential to make an already burgeoning food source markedly more friendly on the environment. And by prioritizing environmental concerns and employing a technique that simply turns off a gene rather than introducing one from a different species, Wargelius and her team may contribute to a shift in how genetic engineering is perceived in Norway, a country with some of the strictest regulations regarding genetically modified organisms on the books.
Scientists develop new technology that gives greater control for managing malaria mosquitoes
17650Keele University, Phy Org, 2021-07-06 19:19:27.
Researchers including a Keele University scientist have engineered an innovative approach to disable highly powerful genetic devices that control harmful insect populations. Dr. Roberto Galizi from Keele's School of Life Sciences was part of a research team that previously developed gene drive technologies that proved highly effective in eliminating populations of mosquitoes in the laboratory, offering a powerful new strategy to prevent deadly vector-borne disease such as malaria. Gene drive elements inserted in the mosquito genome can rapidly spread genetic modifications, such as impairment of fertility, throughout the entire population target by biasing their genetic inheritance after mating with wild insects. The gene drive technologies show great potential for suppressing the mosquito species that transmit malaria with increased power compared to other methods, thanks to their capacity to self-spread through the population. However, this also makes it difficult to retain control of this technology once released. So to combat this, the researchers have now developed an innovative and highly effective technology that allows them to control and even reverse the effects of gene drives.
A genetically encoded anti-CRISPR protein constrains gene drive spread and prevents population suppression
17565C. Taxiarchi, A. Beaghton, N. I. Don, K. Kyrou, M. Gribble, D. Shittu, S. P. Collins, C. L. Beisel, R. Galizi and A. Crisanti, Nature Communications, 12:3977. 2021-06-25 15:00:05.
CRISPR-based gene drives offer promising means to reduce the burden of pests and vector-borne diseases. These techniques consist of releasing genetically modified organisms carrying CRISPR-Cas nucleases designed to bias their inheritance and rapidly propagate desired modifications. Gene drives can be intended to reduce reproductive capacity of harmful insects or spread anti-pathogen effectors through wild populations, even when these confer fitness disadvantages. Technologies capable of halting the spread of gene drives may prove highly valuable in controlling, counteracting, and even reverting their effect on individual organisms as well as entire populations. Here we show engineering and testing of a genetic approach, based on the germline expression of a phage-derived anti-CRISPR protein (AcrIIA4), able to inactivate CRISPR-based gene drives and restore their inheritance to Mendelian rates in the malaria vector Anopheles gambiae. Modeling predictions and cage testing show that a single release of male mosquitoes carrying the AcrIIA4 protein can block the spread of a highly effective suppressive gene drive preventing population collapse of caged malaria mosquitoes.
UC San Diego scientists develop the first CRISPR/Cas9-based gene drive in plants
17563M. Aguilera, UC San Diego News Center, 2021-06-25 14:55:55.
With a goal of breeding resilient crops that are better able to withstand drought and disease, University of California San Diego scientists have developed the first CRISPR-Cas9-based gene drive in plants. While gene drive technology has been developed in insects to help stop the spread of vector-borne diseases such as malaria, researchers in Professor Yunde Zhao's lab, along with colleagues at the Salk Institute for Biological Studies, demonstrated the successful design of a CRISPR-Cas9-based gene drive that cuts and copies genetic elements in Arabidopsis plants. Breaking from the traditional inheritance rules that dictate that offspring acquire genetic materials equally from each parent (Mendelian genetics), the new research uses CRISPR-Cas9 editing to transmit specific, targeted traits from a single parent in subsequent generations. Such genetic engineering could be used in agriculture to help plants defend against diseases to grow more productive crops. The technology also could help fortify plants against the impacts of climate change such as increased drought conditions in a warming world.
Using gene drives to control malaria
17594A. Fell, Daily News, 2021-06-25 14:08:33.
A group of UC scientists led by Greg Lanzaro, professor of pathology, microbiology and immunology in the UC Davis School of Veterinary Medicine, recently completed an analysis of a strategy aimed at eliminating malaria from Africa using genetically engineered mosquitoes. Lanzaro’s lab is part of the UC Irvine Malaria Initiative. The laboratories of Anthony James at UC Irvine and Ethan Bier at UC San Diego engineered mosquitoes with synthetic genes that render them incapable of transmitting the malaria parasite and coupled these genes with a CRISPR-Cas9 gene drive to promote their spread into malaria vector populations in Africa. The idea is to ‘drive’ the new malaria-resistance genes into the mosquito population at a much higher rate than could occur naturally.
Selective inheritance of target genes from only one parent of sexually reproduced F1 progeny in Arabidopsis
17567T. Zhang, M. Mudgett, R. Rambabu, B. Abramson, X. Dai, T. P. Michael and Y. Zhao, Nature Communications, 12:3854. 2021-06-22 15:05:16.
Sexual reproduction constrains progeny to inherit allelic genes from both parents. Selective acquisition of target genes from only one parent in the F1 generation of plants has many potential applications including the elimination of undesired alleles and acceleration of trait stacking. CRISPR/Cas9-based gene drives can generate biased transmission of a preferred allele and convert heterozygotes to homozygotes in insects and mice, but similar strategies have not been implementable in plants because of a lack of efficient homology-directed repair (HDR). Here, we place a gene drive, which consists of cassettes that produce Cas9, guide RNAs (gRNA), and fluorescent markers, into the CRYPTOCHROME 1 (CRY1) gene through CRISPR/Cas9-mediated HDR, resulting in cry1drive lines. After crossing the cry1drive/cry1drive lines to wild type, we observe F1 plants which have DNA at the CRY1 locus from only the cry1drive/cry1drive parent. Moreover, a non-autonomous trans-acting gene drive, in which the gene drive unit and the target gene are located on different chromosomes, converts a heterozygous mutation in the target gene to homozygous. Our results demonstrate that homozygous F1 plants can be obtained through zygotic conversion using a CRISPR/Cas9-based gene drive.
Sustainable Food Production: The Contribution of Genome Editing in Livestock
17590A. Menchaca, Sustainability, 13. 2021-06-21 13:52:55.
This article is focused on the scope and perspectives for the application of this technology, which includes improving production traits, enhancing animal welfare through adaptation and resilience, conferring resistance to infectious diseases, and suppressing pests and invasive species that threaten livestock. The main advantages and concerns that should be overcome by science, policy and people are discussed with the aim that this technology can make a real contribution to our collective future. This review is part of the special issue “Genome Editing in Animal Systems to Support Sustainable Farming and Pest Control”.
Population modification strategies for malaria vector control are uniquely resilient to observed levels of gene drive resistance alleles
17502G. C. Lanzaro, H. M. Sánchez C, T. C. Collier, J. M. Marshall and A. A. James, BioEssays, 2021-06-20 13:46:45.
Cas9/guide RNA (gRNA)-based gene drive systems are expected to play a transformative role in malaria elimination efforts., whether through population modification, in which the drive system contains parasite-refractory genes, or population suppression, in which the drive system induces a severe fitness load resulting in population decline or extinction. DNA sequence polymorphisms representing alternate alleles at gRNA target sites may confer a drive-resistant phenotype in individuals carrying them. Modeling predicts that, for observed levels of SGV at potential target sites and observed rates of de novo DRA formation, population modification strategies are uniquely resilient to DRAs. We conclude that gene drives can succeed when fitness costs incurred by drive-carrying mosquitoes are low enough to prevent strong positive selection for DRAs produced de novo or as part of the SGV and that population modification strategies are less prone to failure due to drive resistance.
Temperature-Inducible Precision Guided Sterile Insect Technique
17447N. P. Kandul, J. Liu and O. S. Akbari, bioRxiv, 2021.06.14.448312. 2021-06-14 13:13:08.
Releases of sterile males are the gold standard for many insect population control programs, and precise sex sorting to remove females prior to male releases is essential to the success of these operations. To advance traditional methods for scaling the generation of sterile males, we previously described a CRISPR-mediated precision-guided sterile insect technique (pgSIT), in which Cas9 and gRNA strains are genetically crossed to generate sterile males for release. While effective at generating F1 sterile males, pgSIT requires a genetic cross between the two parental strains which requires maintenance and sexing of two strains in a factory. Therefore, to further advance pgSIT by removing this crossing step, here we describe a next-generation Temperature-Inducible pgSIT (TI-pgSIT) technology and demonstrate its proof-of-concept in Drosophila melanogaster. Importantly, we were able to develop a true-breeding strain for TI-pgSIT that eliminates the requirement for sex sorting, a feature that may help further automate production at scale.
New CRISPR Tools Can Help Contain Mosquito Disease Transmission
17191Anonymous, labcompare, 2021-06-01 20:26:34.
Scientists have now developed several genetic editing tools that help pave the way to an eventual gene drive designed to stop Culex mosquitoes from spreading disease. As detailed in the journal Nature Communications, Xuechun Feng, Valentino Gantz and their colleagues at Harvard Medical School and National Emerging Infectious Diseases Laboratories developed a Cas9/guide-RNA expression "toolkit" designed for Culex mosquitoes. Since such little attention in genetic engineering has been devoted to Culex mosquitoes, the researchers were required to develop their toolkit from scratch, starting with a careful examination of the Culex genome. While Culex mosquitoes are less problematic in the United States, they are much more of a health risk in Africa and Asia, where they transmit the worm causing filariasis, a disease that can lead to a chronic debilitating condition known as elephantiasis. The researchers also demonstrated that their tools could work in other insects.
Analysis of off-target effects in CRISPR-based gene drives in the human malaria mosquito
17141W. T. Garrood, N. Kranjc, K. Petri, D. Y. Kim, J. A. Guo, A. M. Hammond, I. Morianou, V. Pattanayak, J. K. Joung, A. Crisanti and A. Simoni, Proceedings of the National Academy of Sciences, 118:e2004838117. 2021-05-31 13:24:14.
CRISPR-Cas9 nuclease-based gene drives have been developed toward the aim of control of the human malaria vector Anopheles gambiae. Gene drives are based on an active source of Cas9 nuclease in the germline that promotes super-Mendelian inheritance of the transgene by homology-directed repair (“homing”). Understanding whether CRISPR-induced off-target mutations are generated in Anopheles mosquitoes is an important aspect of risk assessment before any potential field release of this technology. We compared the frequencies and the propensity of off-target events to occur in four different gene-drive strains, including a deliberately promiscuous set-up, using a nongermline restricted promoter for SpCas9 and a guide RNA with many closely related sites (two or more mismatches) across the mosquito genome. Under this scenario we observed off-target mutations at frequencies no greater than 1.42%. We witnessed no evidence that CRISPR-induced off-target mutations were able to accumulate (or drive) in a mosquito population, despite multiple generations’ exposure to the CRISPR-Cas9 nuclease construct. Furthermore, judicious design of the guide RNA used for homing of the CRISPR construct, combined with tight temporal constriction of Cas9 expression to the germline, rendered off-target mutations undetectable. The findings of this study represent an important milestone for the understanding and managing of CRISPR-Cas9 specificity in mosquitoes, and demonstrates that CRISPR off-target editing in the context of a mosquito gene drive can be reduced to minimal levels.All raw amplicon sequencing files have been deposited in the National Center for Biotechnology Information (NCBI) BioProject (accession code PRJNA665154).
Researchers Create New CRISPR Tools to Help Contain Mosquito Disease Transmission
18225M. Aguilera, UC San Diego News Center, 2021-05-28 15:43:14.
Much less genetic engineering has been devoted to Culex genus mosquitoes, which spread devastating afflictions stemming from West Nile virus—the leading cause of mosquito-borne disease in the continental United States—as well as other viruses such as the Japanese encephalitis virus (JEV) and the pathogen causing avian malaria, a threat to Hawaiian birds.University of California San Diego scientists have now developed several genetic editing tools that help pave the way to an eventual gene drive designed to stop Culex mosquitoes from spreading disease. Gene drives are designed to spread modified genes, in this case those that disable the ability to transmit pathogens, throughout the targeted wild population.As detailed in the journal Nature Communications, Xuechun Feng, Valentino Gantz and their colleagues at Harvard Medical School and National Emerging Infectious Diseases Laboratories developed a Cas9/guide-RNA expression “toolkit” designed for Culex mosquitoes. Since such little attention in genetic engineering has been devoted to Culex mosquitoes, the researchers were required to develop their toolkit from scratch, starting with a careful examination of the Culex genome.
New CRISPR tools help contain mosquito disease transmission: Genetics toolkit targets less researched Culex mosquitoes, which transmit West Nile virus and avian malaria.
17145University of California - San Diego, ScienceDaily, 2021-05-28 13:29:00.
Since the onset of the CRISPR genetic editing revolution, scientists have been working to leverage the technology in the development of gene drives that target pathogen-spreading mosquitoes such as Anopheles and Aedes species, which spread malaria, dengue and other life-threatening diseases. Much less genetic engineering has been devoted to Culex genus mosquitoes, which spread devastating afflictions stemming from West Nile virus -- the leading cause of mosquito-borne disease in the continental United States -- as well as other viruses such as the Japanese encephalitis virus (JEV) and the pathogen causing avian malaria, a threat to Hawaiian birds. University of California San Diego scientists have now developed several genetic editing tools that help pave the way to an eventual gene drive designed to stop Culex mosquitoes from spreading disease. Gene drives are designed to spread modified genes, in this case those that disable the ability to transmit pathogens, throughout the targeted wild population.
Suppression of female fertility in Aedes aegypti with a CRISPR-targeted male-sterile mutation
17112J. Chen, J. Luo, Y. Wang, A. S. Gurav, M. Li, O. S. Akbari and C. Montell, Proceedings of the National Academy of Sciences, 118:e2105075118. 2021-05-26 18:33:06.
We introduced and characterized the impact on female fertility of an Ae. aegypti mutation that disrupts a gene that is specifically expressed in testes. We used CRISPR/Cas9 to generate a null mutation in the Ae. aegypti β2-tubulin (B2t) gene, which eliminates male fertility. When we allowed wild-type females to first mate with B2t mutant males, most of the females did not produce progeny even after being subsequently exposed to wild-type males. We also introduced B2t mutant and wild-type males simultaneously with wild-type females and found that a larger number of B2t mutant males relative to the wild-type males was effective in significantly suppressing female fertility. These results raise the possibility of employing B2t sterile males to improve the efficacy of SIT in suppressing populations of Ae. aegypti through repeated releases and thereby reduce the transmission of viruses by these invasive mosquitoes.All study data are included in the main text.
Optimized CRISPR tools and site-directed transgenesis towards gene drive development in Culex quinquefasciatus mosquitoes
17147X. Feng, V. López Del Amo, E. Mameli, M. Lee, A. L. Bishop, N. Perrimon and V. M. Gantz, Nature Communications, 12:2960. 2021-05-20 13:34:20.
Culex mosquitoes are a global vector for multiple human and animal diseases, including West Nile virus, lymphatic filariasis, and avian malaria, posing a constant threat to public health, livestock, companion animals, and endangered birds. While rising insecticide resistance has threatened the control of Culex mosquitoes, advances in CRISPR genome-editing tools have fostered the development of alternative genetic strategies such as gene drive systems to fight disease vectors. However, though gene-drive technology has quickly progressed in other mosquitoes, advances have been lacking in Culex. Here, we develop a Culex-specific Cas9/gRNA expression toolkit and use site-directed homology-based transgenesis to generate and validate a Culex quinquefasciatus Cas9-expressing line. We show that gRNA scaffold variants improve transgenesis efficiency in both Culex quinquefasciatus and Drosophila melanogaster and boost gene-drive performance in the fruit fly. These findings support future technology development to control Culex mosquitoes and provide valuable insight for improving these tools in other species.
Targeting conserved sequences circumvents the evolution of resistance in a viral gene drive against human cytomegalovirus
17054M. Walter, R. Perrone and E. Verdin, Journal of virology, 2021-05-19 13:32:53.
Here, we analyze in cell culture experiments the evolution of resistance in a viral gene drive against human cytomegalovirus. We report that, after an initial invasion of the wildtype population, a drive-resistant population is positively selected over time and outcompetes gene drive viruses. However, we show that targeting evolutionary conserved sequences ensures that drive-resistant viruses acquire long-lasting mutations and are durably attenuated. As a consequence, and even though engineered viruses do not stably persist in the viral population, remaining viruses have a replication defect, leading to a long-term reduction of viral levels. This marks an important step toward developing effective gene drives in herpesviruses, especially for therapeutic applications.<b>Importance</b>The use of defective viruses that interfere with the replication of their infectious parent after co-infecting the same cells - a therapeutic strategy known as viral interference - has recently generated a lot of interest. The CRISPR-based system that we recently reported in herpesviruses represents a novel interfering strategy that causes the conversion of wildtype viruses into new recombinant viruses and drives the native viral population to extinction. In this report, we analyzed how targeted viruses evolved resistance against the technology. Through numerical simulations and cell culture experiments with human cytomegalovirus, we show that, after the initial propagation, a resistant viral population is positively selected and outcompetes engineered viruses over time. We show however that targeting evolutionary conserved sequences ensures that resistant viruses are mutated and attenuated, which leads to a long-term reduction of viral levels. This marks an important step toward the development of novel therapeutic strategies against herpesviruses.
Genetic Technologies for Sustainable Management of Insect Pests and Disease Vectors
17157S. Grilli, R. Galizi and C. Taxiarchi, Sustainability, 13. 2021-05-18 11:00:25.
Recent advancements in genetic and genome editing research, augmented by the discovery of new molecular tools such as CRISPR, have revolutionised the field of genetic engineering by enabling precise site-specific genome modifications with unprecedented ease. These technologies have found a vast range of applications, including the development of novel methods for the control of vector and pest insects. According to their genetic makeup and engineering, these tools can be tuned to impose different grades of impact on the targeted populations. Here, we review some of the most recent genetic control innovations under development, describing their molecular mechanisms and performance, highlighting the sustainability potentials of such interventions.
Genetic Manipulation of Ticks: A Paradigm Shift in Tick and Tick-Borne Diseases Research
17164A. Nuss, A. Sharma and M. Gulia-Nuss, Frontiers in Cellular and Infection Microbiology, 11:7. 2021-05-02 19:18:28.
Ticks are obligate hematophagous arthropods that are distributed worldwide and are one of the most important vectors of pathogens affecting humans and animals. Despite the growing burden of tick-borne diseases, research on ticks has lagged behind other arthropod vectors, such as mosquitoes. This is largely because of challenges in applying functional genomics and genetic tools to the idiosyncrasies unique to tick biology, particularly techniques for stable genetic transformations. CRISPR-Cas9 is transforming non-model organism research; however, successful germline editing has yet to be accomplished in ticks. Here, we review the ancillary methods needed for transgenic tick development and the use of CRISPR/Cas9, the most promising gene-editing approach, for tick genetic transformation.
Systematic identification of plausible pathways to potential harm via problem formulation for investigational releases of a population suppression gene drive to control the human malaria vector Anopheles gambiae in West Africa
16668J. B. Connolly, J. D. Mumford, S. Fuchs, G. Turner, C. Beech, A. R. North and A. Burt, Malaria Journal, 20:170. 2021-03-29 18:49:26.
Population suppression gene drive has been proposed as a strategy for malaria vector control. A CRISPR-Cas9-based transgene homing at the doublesex locus (dsxFCRISPRh) has recently been shown to increase rapidly in frequency in, and suppress, caged laboratory populations of the malaria mosquito vector Anopheles gambiae. Here, problem formulation, an initial step in environmental risk assessment (ERA), was performed for simulated field releases of the dsxFCRISPRh transgene in West Africa.
Driving to Safety: CRISPR-Based Genetic Approaches to Reducing Antibiotic Resistance
16614E. Bier and V. Nizet, Trends in Genetics, 2021-03-18 17:34:35.
The complex and challenging problem of reducing antibiotic resistance (AR) requires a network of both societal and science-based solutions to preserve the most lifesaving pharmaceutical intervention known to medicine. In addition to developing new classes of antibiotics, it is essential to safeguard the clinical efficacy of existing drugs. In this review, we examine the potential application of novel CRISPR-based genetic approaches to reducing AR in both environmental and clinical settings and prolonging the utility of vital antibiotics.
Ultra-conserved sequences in the genomes of highly diverse Anopheles mosquitoes, with implications for malaria vector control
16629S. M. O'Loughlin, A. J. Forster, S. Fuchs, T. Dottorini, T. Nolan, A. Crisanti and A. Burt, G3-Genes Genomes Genetics, 2021-03-18 14:15:51.
Here we search for conserved sequences of 18bp and over in an alignment of 21 Anopheles genomes, spanning an evolutionary timescale of 100 million years, and characterise the resulting sequences according to their location and function. Over 8000 ultra-conserved elements were found across the alignment, with a maximum length of 164 bp. Length-corrected gene ontology analysis revealed that genes containing Anopheles ultra-conserved elements were over-represented in categories with structural or nucleotide binding functions. Known insect transcription factor binding sites were found in 48% of intergenic Anopheles ultra-conserved elements. When we looked at the genome sequences of 1142 wild-caught mosquitoes we found that 15% of the Anopheles ultra-conserved elements contained no polymorphisms. Our list of Anopheles ultra-conserved elements should provide a valuable starting point for the selection and testing of new targets for gene-drive modification in the mosquitoes that transmit malaria.
Meiotic Cas9 expression mediates genotype conversion in the male and female mouse germline.
16618A. J. Weitzel, H. A. Grunwald, R. Levina, V. M. Gantz, S. M. Hedrick, E. Bier and K. L. Cooper, 2021.03.16.435716, 2021-03-17 17:52:21.
We previously showed that such a system of genotype conversion from heterozygous to homozygous after a sequence targeted CRISPR/Cas9 double strand DNA break is feasible in the female mouse germline. In the male germline, however, all double strand breaks were instead repaired by end joining mechanisms to form an 'insertion/deletion' (indel) mutation. These observations suggested that timing Cas9 expression to coincide with meiosis I is critical to favor conditions when homologous chromosomes are aligned and interchromosomal homology directed repair (HDR) mechanisms predominate. Here, using a Cas9 knock-in allele at the Spo11 locus, we show that meiotic expression of Cas9 does indeed mediate genotype conversion in the male as well as in the female germline. However, the low frequency of both HDR and indel mutation in both male and female germlines suggests that Cas9 may be expressed from the Spo11 locus at levels too low for efficient double strand DNA break formation. We suggest that more robust Cas9 expression initiated during early meiosis I may improve the efficiency of genotype conversion and further increase the rate of 'super-Mendelian' inheritance from both male and female mice.Competing Interest StatementVMG, SMH, EB, and KLC hold advisory board positions with Synbal, Inc. All other authors declare that they have no competing interests.
Genetically Encoded CRISPR components Yield Efficient Gene Editing in the Invasive Pest, Drosophila suzukii
16602N. P. Kandul, E. J. Belikoff, J. Liu, A. Buchman, F. Li, A. Yamamoto, T. Yang, I. Shriner, M. J. Scott and O. Akbari, bioRxiv, 2021.03.15.435483. 2021-03-16 18:28:51.
Here we have developed transgenic strains that encode three different terminators and four different promoters to express Cas9 in both the soma and/or germline of SWD. The Cas9 lines were evaluated through genetic crossing to transgenic lines that encode single guide RNAs targeting the conserved X-linked yellow body and white eye genes. We find that several Cas9/gRNA lines display very high editing capacity. Going forward, these tools will be instrumental for evaluating gene function in SWD and may provide tools useful for the development of new genetic strategies for control of this invasive species.
Gene Drives Built to Follow More Stringent Rules of the Road
16590Anonymous, Genetic Engineering & Biotechnology News, 2021-03-08 19:57:32.
Gene drives, or systems that accelerate the spread of desirable genetic traits into a population, may be built to achieve specific levels of spread when released into the wild. By exerting control over the degree of spread, those who unleash gene drives may realize the benefits promised by gene drives—the suppression of disease-carrying or crop-destroying insects—while minimizing the risks that unintended genetic changes could occur that would lead to undesirable ecological outcomes. A gene drive engineered to allow for a high degree of control was recently introduced by scientists based at the University of California, San Diego (UCSD). The new gene drive is a “split drive” system. Details appeared in an article titled, “Inherently confinable split-drive systems in Drosophila.” The article demonstrates that split-drive systems may allow various genetic parameters and strategies to be used to either limit or extend drive potential.
New gene-drive technologies can help control crop pests
16569Anonymous, AZO Life Sciences, 2021-03-08 14:21:28.
The supposed gene drives, which exploit CRISPR technology to affect genetic inheritance, show the potential to quickly spread particular genetic traits across the populations of a specified species. For instance, gene-drive technologies used on insects are being developed to stop the spread of devastating diseases, like dengue and malaria, by inhibiting mosquito hosts from becoming infected. Gene-drives are being designed in agricultural fields to help regulate or remove economically damaging crop pests. But together with the potential to modify populations, there have been concerns about the long-term impacts of these transformative novel technologies in the wild. Both scientists and ethicists have queried about how the so-called gene drives, once turned loose in a regional population, can be controlled if required. Scientists from the University of California San Diego (UC San Diego), Tata Institute for Genetics and Society (TIGS) at UC San Diego, and their collaborators at UC Berkeley have now designed a new technique that gives more control over the release of gene drives. Information about the new “split drive” was published in the Nature Communications and eLife journals on March 5th, 2021.
New ‘Split-drive’ System Puts Scientists in the (Gene) Driver Seat
16555M. Aguilera, UC San Diego News Center, 2021-03-05 19:42:37.
Gene-drive technologies applied in insects, for example, are being designed to halt the spread of devastating diseases such as malaria and dengue by preventing mosquito hosts from becoming infected. In agricultural fields, gene-drives are being developed to help control or eliminate economically damaging crop pests. But along with the capacity to alter populations, concerns have been raised regarding the long-term effects of these transformative new technologies in the wild. Researchers and ethicists have voiced questions about how gene drives, once turned loose in a regional population, could be held in check if necessary. Now, researchers at the University of California San Diego, Tata Institute for Genetics and Society (TIGS) at UC San Diego and their colleagues at UC Berkeley have developed a new method that provides more control over gene drive releases. Details of the new “split drive” are published March 5 in the journals Nature Communications and eLife.
A confinable home and rescue gene drive for population modification
16579N. P. Kandul, J. Liu, J. B. Bennett, J. M. Marshall and O. S. Akbari, eLife, 10:e65939. 2021-03-05 16:30:16.
Homing based gene drives, engineered using CRISPR/Cas9, have been proposed to spread desirable genes throughout populations. However, invasion of such drives can be hindered by the accumulation of resistant alleles. To limit this obstacle, we engineer a confinable population modification Home-and-Rescue (HomeR) drive in Drosophila targeting an essential gene. In our experiments, resistant alleles that disrupt the target gene function were recessive lethal, and therefore disadvantaged. We demonstrate that HomeR can achieve an increase in frequency in population cage experiments, but that fitness costs due to the Cas9 insertion limit drive efficacy.
Developing GDi-CRISPR System for Multi-copy Integration in Saccharomyces cerevisiae
16544Z.-X. Zhang, Y.-Z. Wang, Y.-S. Xu, X.-M. Sun and H. Huang, Applied Biochemistry and Biotechnology, 2021-03-03 16:12:03.
This study aims to develop a low-cost and easy-to-use multi-copy integration tool in S. cerevisiae. Firstly, twenty-one Cas proteins from different microorganisms were tested in S. cerevisiae to find the functional Cas proteins with optimal cleavage ability. Results showed that eight Cas proteins can complete gene editing. However, most of the transformants have low copy numbers, which may be caused by high cutting efficiency exceeding the repair rate. Therefore, the effect of donor translocation order was further investigated. Results showed that 4 copies were obtained by donor first translocation. Then, the gene drive delta site integration system by the CRISPR system (GDi-CRISPR) was developed by combining gene drive principle and CRISPR system. To be clear, the gRNA was put into donor fragments. Then, both of them were integrated into the genome, which can drive further cutting and repair due to increasing number of gRNA. Instead of high-throughput screening or resistance pressure, 6 copies were obtained in only 5–6 days using the GDi-CRISPR system. It is expected to further advance the development of S. cerevisiae multi-copy integration tools.
Designing gene drives to limit spillover to non-target populations
16516G. Greenbaum, M. W. Feldman, N. A. Rosenberg and J. Kim, PLOS Genetics, 17:e1009278. 2021-02-25 15:02:27.
We develop mathematical models of gene-drive dynamics that incorporate migration between a target and non-target populations to investigate the possibility of effectively applying a gene drive in the target population while limiting its spillovers to the non-target population (‘differential targeting’). We observe that the feasibility of differential targeting depends on the gene-drive design specification, as well as on the migration rates between the populations. Even when differential targeting is possible, as migration increases, the possibility for differential targeting disappears. We find that differential targeting can be effective for low migration rates, and that it is sensitive to the design of the gene drive under high migration rates. We suggest that differential targeting could be used, in combination with other mitigation measures, as an additional safeguard to limit gene drive spillovers.
Population genomics of invasive rodents on islands: Genetic consequences of colonization and prospects for localized synthetic gene drive
16657K. P. Oh, A. B. Shiels, L. Shiels, D. V. Blondel, K. J. Campbell, J. R. Saah, A. L. Lloyd, P. Q. Thomas, F. Gould, Z. Abdo, J. R. Godwin and A. J. Piaggio, Evolutionary Applications, 2021-02-22 17:41:41.
Here we used pooled whole-genome sequencing of invasive mouse (Mus musculus) populations on four islands along with paired putative source populations to test genetic predictions of island colonization and characterize locally fixed Cas9 genomic targets. Patterns of variation across the genome reflected marked reductions in allelic diversity in island populations and moderate to high degrees of differentiation from nearby source populations despite relatively recent colonization. Locally fixed Cas9 sites in female fertility genes were observed in all island populations, including a small number with multiplexing potential. In practice, rigorous sampling of presumptive LFA will be essential to fully assess risk of resistance alleles. These results should serve to guide development of improved, spatially limited gene drive design in future applications.
Split versions of Cleave and Rescue selfish genetic elements for measured self limiting gene drive
16426G. Oberhofer, T. Ivy and B. A. Hay, PLoS genetics, 17:e1009385. 2021-02-18 20:00:28.
Self-sustaining Cleave and Rescue (ClvR) elements include a DNA sequence-modifying enzyme such as Cas9/gRNAs that disrupts endogenous versions of an essential gene, a tightly linked recoded version of the essential gene resistant to cleavage (the Rescue), and a Cargo. ClvR spreads by creating loss-of-function (LOF) conditions in which those without ClvR die because they lack functional copies of the essential gene. We use modeling to show that when the Rescue-Cargo and one or both components required for LOF allele creation (Cas9 and gRNA) reside at different locations (split ClvR), drive of Rescue-Cargo is self-limiting due to a progressive decrease in Cas9 frequency, and thus opportunities for creation of LOF alleles, as spread occurs. Importantly, drive strength and duration can be extended in a measured manner-which is still self-limiting-by moving the two components close enough to each other that they experience some degree of linkage. With linkage, Cas9 transiently experiences drive by hitchhiking with Rescue-Cargo until linkage disequilibrium between the two disappears, a function of recombination frequency and number of generations, creating a novel point of control. We implement split ClvR in Drosophila, with key elements on different chromosomes. Cargo/Rescue/gRNAs spreads to high frequency in a Cas9-dependent manner, while the frequency of Cas9 decreases.
Emergent challenges for CRISPR: biosafety, biosecurity, patenting, and regulatory issues
19398Braddick, D. , and Ramarohetra, R. F., Genome Engineering Via Crispr-Cas9 System, 2021-02-18 17:46:33.
The recent advancements of CRISPR-Cas technologies have transformed this simple and efficient gene editing technique into an extraordinarily powerful tool. The most anticipated applications could create novel therapeutics against mankind's most serious afflictions and help eradicate vector-based diseases. However, with these desired benefits come new ethical questions and potential threats. CRISPR technologies are not yet fully developed toward delivering all of their promises, and suffer from biosafety problems in human products and biosecurity threats arising from its potential dual use and errant gene drives. Furthermore, the future innovations of CRISPR technologies may encounter non-scientific challenges in patentability and unclear legal regulations at the national and international levels. This chapter will discuss these issues, and where possible, will highlight the currently proposed solutions that could mitigate the threats, and could address the problems that hold back CRISPR's full potential.
Scientifically framed gene drive communication perceived as credible but riskier
16406E. A. MacDonald, E. D. Edwards, J. Balanovic and F. Medvecky, People and Nature, 2021-02-17 18:23:29.
Framing is a communication technique in which certain beliefs or values are emphasized that resonate with the target audience. Framing may increase how much people objectively think about new information and update their opinions; framing may mitigate emo
ReMOT Control Delivery of CRISPR-Cas9 Ribonucleoprotein Complex to Induce Germline Mutagenesis in the Disease Vector Mosquitoes Culex pipiens pallens (Diptera: Culicidae)
17482X. X. Li, Y. Xu, H. B. Zhang, H. T. Yin, D. Zhou, Y. Sun, L. Ma, B. Shen and C. L. Zhu, Journal of Medical Entomology, 58. 2021-02-16 14:57:35.
The wide distribution of Culex (Cx.) pipiens complex mosquitoes makes it difficult to prevent the transmission of mosquito-borne diseases in humans. Gene editing using CRISPR/Cas9 is an effective technique with the potential to solve the growing problem of mosquito-borne diseases. This study uses the ReMOT Control technique in Culex pipiens pallens (L.) to produce genetically modified mosquitoes. A microinjection system was established by injecting 60 adult female mosquitoes-14 mu l injection mixture was required, and no precipitation occurred with <= 1 mu l of endosomal release reagents (chloroquine or saponin). The efficiency of delivery of the P2C-enhanced green fluorescent protein-Cas9 (P2C-EGFP-Cas9) ribonucleoprotein complex into the ovary was 100% when injected at 24 h post-bloodmeal (the peak of vitellogenesis). Using this method for KMO knockout, we found that gene editing in the ovary could also occur when P2C-Cas9 RNP complex was injected into the hemolymph of adult Cx. pipiens pallens by ReMOT Control. In the chloroquine group, of the 2,251 G(0) progeny screened, 9 individuals showed with white and mosaic eye phenotypes. In the saponin group, of the 2,462 G(0) progeny screened, 8 mutant individuals were observed. Sequencing results showed 13 bp deletions, further confirming the fact that gene editing occurred. In conclusion, the successful application of ReMOT Control in Cx. pipiens pallens not only provides the basic parameters (injection parameters and injection time) for this method but also facilitates the study of mosquito biology and control.
Optimized CRISPR tools and site-directed transgenesis in Culex quinquefasciatus mosquitoes for gene drive development
16372X. Feng, V. Lopez Del Amo, E. Mameli, M. Lee, A. L. Bishop, N. Perrimon and V. M. Gantz, bioRxiv, 2021.02.10.430702. 2021-02-11 20:33:00.
Here, we developed a Culex-specific Cas9/gRNA expression toolkit and used site-directed homology-based transgenesis to generate and validate a Culex quinquefasciatus Cas9-expressing line. We showed that gRNA scaffold variants improve transgenesis efficiency in both Culex and Drosophila and boost gene-drive performance in the fruit fly. These findings support future technology development to control Culex mosquitoes and provide valuable insight for improving these tools in other species.
A Code of Ethics for Gene Drive Research
16379G. J. Annas, C. L. Beisel, K. Clement, A. Crisanti, S. Francis, M. Galardini, R. Galizi, J. Grünewald, G. Immobile, A. S. Khalil, R. Müller, V. Pattanayak, K. Petri, L. Paul, L. Pinello, A. Simoni, C. Taxiarchi and J. K. Joung, The CRISPR Journal, 4:19:1-8. 2021-02-10 14:57:13.
A code of ethics can be a useful tool for all parties involved in the development and regulation of gene drives and can be used to help ensure that a balanced analysis of risks, benefits, and values is taken into consideration for the interest of society and humanity. We have developed a code of ethics for gene drive research with the hope that this code will encourage the development of an international framework that includes ethical guidance of gene drive research and is incorporated into scientific practice by gaining broad agreement and adherence.
Assisting Evolution: How Far Should We Go to Help Species Adapt?
16369E. Kolbert, YaleEnvironment360, 2021-02-09 20:28:55.
It was a hot, intensely blue day in the Australian Outback, about 350 miles north of Adelaide. I was tagging along with Moseby as she checked the batteries on the motion-sensitive cameras that dot Arid Recovery, an ecosystem restoration project she and her husband launched in 1997. The project sprawls over 47 square miles of red earth and scrub. It’s entirely surrounded by a six-foot-tall fence, which is designed to keep out feral cats and foxes. Inside the main fence is a series of smaller fenced-in paddocks. Several years ago, Moseby decided to start adding cats into some of these. Her reasoning was simple and, in its own way, radical. The outback ecosystem had been so fundamentally changed, that, if the native animals were to survive, they would have to change, too. Perhaps they could be trained to avoid cats, which were introduced to the country by the British colonists and now can be found virtually everywhere in Australia, including most islands.
Should we dim the sun? Will we even have a choice
16384E. Klein, New York Times, 2021-02-09 15:06:18.
“Under a White Sky” is going to be on my best books of 2021 list. It’s a wonderful work. Kolbert is the Pulitzer Prize-winning author of “The Sixth Extinction,” which you may have read. She is a staff writer at The New Yorker and just one of the great science journalists of this time, and particularly one of the great climate journalists of this age. But this book, this book’s existence is evidence of how badly that fight is going. This is a book about what we are going to need to contemplate in the coming years that we don’t want to. It’s a book about taking responsibility for how irreversibly we have altered the natural world; how often we have tried to control it, and then watched those attempts at control fail; how often the best most scientific minds of the age have come up with some brilliant solution, implemented it, and then watched calamity result. And at the same time — and this is what makes the book so worthwhile — it is a book about how there is no going back. Not now, not ever. We are in the Anthropocene. The future from here is an endless layering on of new efforts to control the consequences of our past efforts. We don’t get to flinch or pretend we don’t have to contemplate any of this. We’ve gone too far. One of the hardest things to do as a writer — and I tell you this from personal experience — is to write ambivalence. It’s easy to write a polemic or a sharp take. It is hard to write down the middle path, where you are simply describing things as they are, knowing that every possible obvious answer you can come to is probably a bad one, knowing that the hubris embedded in past attempts to solve this problem means any future brilliant idea is likely to end that way, too, but that doesn’t mean we can do nothing. But Kolbert walks that path really beautifully here, which is why I wanted to talk to her for the show. As always, my email is [email protected]. I’m always interested to know who you’d like to see on the show. The weirder, the better. So send me your guest suggestions. Here’s Elizabeth Kolbert.
In Our Image: The Ethics of CRISPR Genome Editing
16272J. C. Eissenberg, Biomolecular Concepts, 12:1-7. 2021-02-06 16:55:38.
Here, I discuss the ethics surrounding the transformative CRISPR/Cas9mediated genome editing technology in the contexts of human genome editing to eradicate genetic disease and of gene drive technology to eradicate animal vectors of human disease.
CRISPR and the splice to survive: New gene-editing technology could be used to save species from extinction—or to eliminate them.
16011E. Kolbert, New Yorker, 2021-01-11 14:25:59.
About a year ago, not long before the pandemic began, I paid a visit to the center, which is an hour southwest of Melbourne. The draw was an experiment on a species of giant toad known familiarly as the cane toad. The toad was introduced to Australia as an agent of pest control, but it promptly got out of control itself, producing an ecological disaster. Researchers at the A.C.D.P. were hoping to put the toad back in the bottle, as it were, using crispr.
Targeting evolutionary conserved sequences circumvents the evolution of resistance in a viral gene drive against human cytomegalovirus
15943M. Walter, R. Perrone and E. Verdin, bioRxiv, 2021.01.08.425902. 2021-01-08 16:26:29.
Here, we analyze in cell culture experiments the evolution of resistance in a gene drive against human cytomegalovirus. We report that after an initial invasion of the wildtype population, a drive-resistant population is positively selected over time and outcompetes gene drive viruses. However, we show that targeting evolutionary conserved regions ensures that drive-resistant viruses have a replication defect, leading to a long-term reduction of viral levels. This marks an important step toward developing effective gene drives in viruses, especially for therapeutic applications.
Control of malaria-transmitting mosquitoes using gene drives
15693T. Nolan, Philosophical Transactions of the Royal Society B: Biological Sciences, 376:20190803. 2020-12-28 15:31:23.
In this article, I will discuss the relative merits of this type of gene drive, as well as barriers to its technical development and to its deployment in the field as malaria control. This article is part of the theme issue ‘Novel control strategies for mosquito-borne diseases'.
Vector dynamics influence spatially imperfect genetic interventions against disease
16547M. K. Yuksel, C. H. Remien, B. Karki, J. J. Bull and S. M. Krone, Evolution, Medicine, and Public Health, 9:1-10. 2020-12-27 16:20:40.
In spatially structured populations, imperfect coverage of the vector will leave pockets in which the parasite may persist. Movement by humans may disrupt this local persistence and facilitate eradication when these pockets are small, spreading parasite reproduction outside unprotected areas and into areas that block its reproduction. Here, we consider the sensitivity of this process to biological details: do simple generalities emerge that may facilitate interventions?We develop formal mathematical models of this process similar to standard Ross–Macdonald models, but (i) specifying spatial structure of two patches, with vector transmission blocked in one patch but not in the other, (ii) allowing temporary human movement (travel instead of migration) and (iii) considering two different modes of mosquito biting.We find that there is no invariant effect of disrupting spatial structure with travel.
New GE unintentionally leaves traces in cells
15620C. Then, Testbiotech, 2020-12-18 16:22:58.
A new scientific publication shows that CRISPR/Cas gene scissor applications in animals unintentionally leave traces. The findings are not related to unintended changes in the DNA, which have often been described, but to gene regulation, i.e. epigenetics. The effects are heritable and may, for example, result in disruption of embryonic development. The new scientific publication describes CRISPR/Cas experiments with mice in which their DNA is cut and additional genetic information inserted. Besides intended changes in DNA in the target region, the findings also showed unintended changes in so-called epigenetic markers that control gene regulation. The effects were heritable and could still be identified after ten generations. According to the authors, the effects can also be used to identify CRISPR/Cas gene scissor applications.
New insect species made via genetic engineering
15610L. Leffer, SCIENCELINE, 2020-12-18 15:47:16.
A biotech fast-forward button for evolution is on the horizon. Researchers say they have used a novel genetic engineering method to create several new species of fruit fly in the lab for the first time — an achievement which might help put a future without malaria and other insect-borne diseases within reach. The approach, called synthetic speciation, could prove useful in creating safer pest-control technologies, says Maciej Maselko, a postdoctoral fellow studying synthetic biology at Macquarie University. In one far-off scenario, according to Maselko, synthetic speciation might even be applied to generate designer organisms that could pollinate plants or even detect landmines. Maselko and his team published their findings September 8 in Nature Communications. “Speciation has occurred billions of times on the planet, but hasn’t been engineerable [before],” says Michael Smanski, a molecular biologist at the University of Minnesota and member of the research team. Maselko, Smanski and their colleagues have previously used a similar method to engineer “species like” differences in yeast in 2018, but their more recent results are the first time the concept has been proven possible in a multicellular animal. This method could produce untold numbers of new animal varieties within months rather than millennia, Smanski says.
A CRISPR endonuclease gene drive reveals two distinct mechanisms of inheritance bias
15480S. A. N. Verkuijl, E. González, J. X. D. Ang, M. Li, N. P. Kandul, M. Anderson, O. S. Akbari, M. Bonsall and L. Alphey, bioRxiv, 2020.12.15.421271. 2020-12-16 14:45:08.
In this study, we report the functioning of sds3, bgcn, and nup50 expressed Cas9 in an Aedes aegypti homing split drive system targeting the white gene. We report their inheritance biasing capability, propensity for maternal deposition, and zygotic/somatic expression. Additionally, by making use of the tight linkage of white to the sex-determining locus, we were able to elucidate mechanisms of inheritance bias. We find inheritance bias through homing in double heterozygous males, but find that a previous report of the same drive occurred through meiotic drive. We propose that other previously reported 'homing'design gene drives may in fact bias their inheritance through other mechanisms with important implications for gene drive design.Competing Interest StatementThe authors have declared no competing interest.
The Antiviral Small-Interfering RNA Pathway Induces Zika Virus Resistance in Transgenic Aedes aegypti
15482A. E. Williams, I. Sanchez-Vargas, W. R. Reid, J. Y. Lin, A. W. E. Franz and K. E. Olson, Viruses, 12:18. 2020-12-15 14:45:29.
We used CRISPR/Cas9 to re-target a previously characterized locus (Chr2:321382225) and engineered mosquitoes expressing an inverted repeat (IR) dsRNA against the NS3/4A region of the ZIKV genome. Small RNA analysis revealed that the IR effector triggered the mosquito's siRNA antiviral pathway in bloodfed females. Nearly complete (90%) inhibition of ZIKV replication was found in vivo in both midguts and carcasses at 7 or 14 days post-infection (dpi). Furthermore, significantly fewer transgenic mosquitoes contained ZIKV in their salivary glands (p = 0.001), which led to a reduction in the number of ZIKV-containing saliva samples as measured by transmission assay. Our work shows that Ae. aegypti innate immunity can be co-opted to engineer mosquitoes resistant to ZIKV.
Evading resistance to gene drives
15422R. Gomulkiewicz, M. L. Thies and J. J. Bull, bioRxiv, 2020.08.27.270611. 2020-12-12 15:44:15.
Our analyses suggest that among gene drives that cause moderate suppression, toxin-antidote systems are less apt to select for resistance than homing drives. Single drives of moderate effect might cause only moderate population suppression, but multiple drives (perhaps delivered sequentially) would allow arbitrary levels of suppression. The most favorable case for evolution of resistance appears to be with suppression homing drives in which resistance is dominant and fully suppresses transmission distortion; partial suppression by resistance heterozygotes or recessive resistance are less prone to resistance evolution. Given that it is now possible to engineer CRISPR-based gene drives capable of circumventing allelic resistance, this design may allow for the engineering of suppression gene drives that are effectively resistance-proof.
Mosquito population modification: the drive to malaria eradication
15272A. A. James, BugBitten BMC, 2020-11-27 14:37:56.
We have had considerable success in the past demonstrating that we can use modern molecular biological and insect transgenesis tools to make genes that prevent mosquitoes from passing on parasites (see 1 and 2). We have focused most recently on laboratory experiments to find ways to move these genes into wild mosquito populations.
Split drive killer-rescue provides a novel threshold-dependent gene drive
15254M. P. Edgington, T. Harvey-Samuel and L. Alphey, Scientific Reports, 10. 2020-11-25 18:32:01.
Population genetics mathematical models are developed here to demonstrate the threshold-dependent nature of the proposed system and its robustness to imperfect homing, incomplete penetrance of toxins and transgene fitness costs, each of which are of practical significance given that real-world components inevitably have such imperfections. We show that although end-joining repair mechanisms may cause the system to break down, under certain conditions, it should persist over time scales relevant for genetic control programs. The potential of such a system to provide localised population suppression via sex ratio distortion or female-specific lethality is also explored. Additionally, we investigate the effect on introduction thresholds of adding an extra CRISPR base element, showing that this may either increase or decrease dependent on parameter context
Gene drive blocks malaria transmission in mosquitoes
15035labonline, labonline, 2020-11-09 15:46:59.
Employing a strategy known as ‘population modification’, which involves using a CRISPR-Cas9 gene drive system to introduce genes preventing parasite transmission into mosquito chromosomes, University of California (UC) researchers have made a major advance in the use of genetic technologies to control the transmission of malaria parasites. Their work has been published in the journal Nature Communications.
A gene-drive rescue system for the modification of malaria mosquito populations
14901A. Adolfi, Nature Research Bioengineering Community, 2020-11-03 18:48:29.
Mosquito populations can now be reliably modified using 1) antimalarial molecules that block parasite development and 2) a CRISPR-based gene drive system that mediates their rapid spreading across the vector population.
UC researchers pioneer more effective method of blocking malaria transmission in mosquitoes
14898UCI, UCI News, 2020-11-03 14:11:01.
University of California, Irvine postdoctoral researcher Adriana Adolfi, in collaboration with colleagues at UCI, UC Berkeley and UC San Diego, followed up on the group’s pioneering effort to develop CRISPR-based gene drive systems for making mosquito vectors resistant to transmitting malaria parasites by increasing gene drive effectiveness in female mosquito progeny.The second-generation gene drive system described in this paper can be applied to any of the several thousand genes that are essential for insects to survive or reproduce,” said UC San Diego Distinguished Professor Ethan Bier, a co-author of the study and science director at the Tata Institute for Genetics and Society. “While it was developed in fruit flies, this system is readily transportable to a broad selection of insect species that serve as vectors for devastating disorders such as Chagas disease, sleeping disease, leishmaniasis and arboviral diseases.”
Gene Drive: The What, How, Why, and Whether We Should
14865N. Pazhayam, The Pipettepen, 2020-10-26 19:12:07.
Under regular Mendelian inheritance, the probability of inheriting a particular allele from a heterozygous parent is 50% – this is because offspring can only inherit one or the other chromosome from each parent. However, gene drive is a technology that changes this probability and makes it much higher than 50%.
WHO Refers to GM Mosquitoes as Beneficial Technology
14820ISAAA, Crop Biotech Update, 2020-10-21 17:27:43.
The World Health Organization (WHO) released its official statement to clarify its stance on the evaluation and use of genetically modified (GM) mosquitoes and its use to control vector-borne diseases (VBD). WHO says it supports the investigation of all potentially beneficial technologies, and these include GM mosquitoes.
Position of ARRIGE Scientific Committee on Gene Drive
14801ARRIGE Scientific Committee on Gene Drive, ARRIGE Newsletter, 2020-10-21 15:16:40.
We are facing a change of paradigm that must lead us to be responsible for altered inheritance and the hybridization between artefacts, considered as natural or artificial, at the very moment when this division itself is blurred by the engineering capacity to act on mutations, without knowing in advance the possibly adverse, short or long term, side effects, on the manipulated and other connected species.
Progress Toward Zygotic and Germline Gene Drives in Mice
14852C. Pfitzner, M. A. White, S. G. Piltz, M. Scherer, F. Adikusuma, J. N. Hughes and P. Q. Thomas, The CRISPR Journal, 3:388-397. 2020-10-20 17:17:24.
Here, we investigated the efficiency of CRISPR-Cas9-based gene drives in Mus musculus by constructing "split drive" systems where gRNA expression occurs on a separate chromosome to Cas9, which is under the control of either a zygotic (CAG) or germline (Vasa) promoter.
GeneConvene Global Collaborative Webinar Series | Gene Drive Technical Webinars
14347David O'Brochta and Hector Quemada, GeneConvene Global Collaborative, 2020-10-12 02:54:40.
A series of technical webinars on engineered gene drive technology research and development given by leading researchers in the field.
Gene Drives Could Kill Mosquitoes And Suppress Herpesvirus Infections
14588A. Berezow, American Council on Science and Health, 2020-09-30 20:03:47.
A team of researchers writing in the journal Nature Communications has shown that a gene drive can be used to suppress infection with cytomegalovirus, a type of herpesvirus.
Resistance to natural and synthetic gene drive systems
14468T. A. R. Price, N. Windbichler, R. L. Unckless, A. Sutter, J.-N. Runge, P. A. Ross, A. Pomiankowski, N. L. Nuckolls, C. Montchamp-Moreau, N. Mideo, O. Y. Martin, A. Manser, M. Legros, A. M. Larracuente, L. Holman, J. Godwin, N. Gemmell, C. Courret, A. Buc, Journal of Evolutionary Biology, 2020-09-24 17:20:31.
This review summarizes our current knowledge of drive resistance in both natural and synthetic gene drives. We explore how insights from naturally occurring and synthetic drive systems can be integrated to improve the design of gene drives, better predict the outcome of releases and understand genomic conflict in genera
Inherently confinable split-drive systems in Drosophila
14294G. Terradas, A. B. Buchman, J. B. Bennett, I. Shriner, J. M. Marshall, O. S. Akbari and E. Bier, bioRxiv, 2020.09.03.282079. 2020-09-03 14:16:46.
Here, we test split gene-drive (sGD) systems in Drosophila melanogaster that were inserted into essential genes required for viability (rab5, rab11, prosalpha2) or fertility (spo11). I
Chromosome drives via CRISPR-Cas9 in yeast
15025H. Xu, M. Han, S. Zhou, B.-Z. Li, Y. Wu and Y.-J. Yuan, Nature Communications, 11:4344. 2020-08-28 15:22:50.
Our results show that the entire Saccharomyces cerevisiae chromosome can be eliminated efficiently through only one double-strand break around the centromere via CRISPR-Cas9. As a proof-of-concept experiment of this CRISPR-Cas9 chromosome drive system, the synthetic yeast chromosome X is completely eliminated, and the counterpart wild-type chromosome X harboring a green fluorescent protein gene or the components of a synthetic violacein pathway are duplicated by sexual reproduction. We also demonstrate the use of chromosome drive to preferentially transmit complex genetic traits in yeast.
Evading evolution of resistance to gene drives
14174R. Gomulkiewicz, M. L. Thies and J. J. Bull, bioRxiv, 2020-08-27 14:29:17.
Here we develop mathematical and computational models to identify conditions under which suppression drives will evade resistance, even if resistance is present initially.
Next-generation gene drive for population modification of the malaria vector mosquito, Anopheles gambiae
14015R. Carballar-Lejarazú, C. Ogaugwu, T. Tushar, A. Kelsey, T. B. Pham, J. Murphy, H. Schmidt, Y. Lee, G. C. Lanzaro and A. A. James, Proceedings of the National Academy of Sciences, 202010214. 2020-08-24 12:38:12.
We show here that the Cas9/guide RNA-based gene-drive components of a genetically-engineered malaria mosquito vector, Anopheles gambiae, achieve key target product profile requirements for efficacy and performance.
A home and rescue gene drive forces its inheritance stably persisting in populations
14006N. P. Kandul, J. Liu, J. B. Bennett, J. M. Marshall and O. Akbari, bioRxiv, 2020.08.21.261610. 2020-08-23 17:46:58.
We demonstrate that HomeR can achieve nearly ~100% transmission enabling it to persist at genotypic fixation in several multi-generational population cage experiments, underscoring its long term stability.
Towards Responsive Eco-technology: The Development of a Male Sex-biased Mouse
24607W. Kamau, Massachusetts Institute of Technology, 2020-08-16 08:55:45.
CRISPR-Cas systems have catalyzed the emergence of several synthetic population management strategies, like gene drives, for controlling pests and disease vectors. As these technologies garner greater visibility in both general and regulatory audiences, questions have arisen about the invasiveness of drive strategies and have underscored a need for community guidance in designing population management technologies. In heterogametic species, an engineered male-determining chromosome can serve as a method for providing robust and localized population suppression without the need for a gene drive. In mice, X-chromosome inactivation is mediated by X-inactive specific transcript (Xist) long non-coding RNA. I propose to encode a system on the Y chromosome to knock out a necessary region for proper X-inactivation in females. Loss of Xist gene function has no known effects in males or females with a dysfunctional maternal copy, however, females who inherit a dysfunctional paternal copy die at embryonic day 8.5. Thus, this results in a male sex-biased mouse. To create a daughterless mouse, my proof-of-principle design will include a constitutively expressed Cas protein with at minimum a two-guide array. Additionally, I will draw on the ecological species concept found in some cultures, like the M¯aori of New Zealand, to create an alternate eco-cisgenic version using cisgenic murine elements and a CRISPR system found in a commensal species of bacteria. Creating a cisgenic non-driving mammalian model of a genetic population suppression system would be a first-of-its-kind example to show how biological engineering design decisions can be congruent with culturally specific notions of ecology.
Modelling the suppression of a malaria vector using a CRISPR-Cas9 gene drive to reduce female fertility
13724A. R. North, A. Burt and H. C. J. Godfray, BMC Biology, 18:98. 2020-08-11 12:23:41.
Gene drives based on CRISPR-Cas9 technology are increasingly being considered as tools for reducing the capacity of mosquito populations to transmit malaria, and one of the most promising options is driving endonuclease genes that reduce the fertility of female mosquitoes. Here, we use simulation modelling to understand the factors affecting the spread of this type of gene drive over a one million-square kilometre area of West Africa containing substantial environmental and social heterogeneity.
Efficient population modification gene-drive rescue system in the malaria mosquito Anopheles stephensi
13593A. Adolfi, V. M. Gantz, N. Jasinskiene, H.-F. Lee, K. Hwang, E. A. Bulger, A. Ramaiah, J. B. Bennett, G. Terradas, J. J. Emerson, J. M. Marshall, E. Bier and A. A. James, bioRxiv, 2020.08.02.233056. 2020-08-02 12:59:26.
We developed the first recoded gene-drive rescue system for population modification in the malaria vector, Anopheles stephensi, that relieves the load in females caused by integration of the drive into the kynurenine hydroxylase gene by rescuing its function. Non-functional resistant alleles are eliminated via a dominantly-acting maternal effect combined with slower-acting standard negative selection, and a functional resistant allele does not prevent drive invasion.
Meet Cosmo, the gene-edited Crispr calf
13590S. D. McClain, Capital Press, 2020-07-31 12:56:28.
Cosmo was the grand finale of a series of experiments to create a line of genome-edited cattle tailored for the beef industry. He was designed to produce 75% male offspring.
The future of beef might be a sausage fest
13479N. Johnson, grist, 2020-07-24 21:00:33.
N. Johnson. (2020) grist. A media report on the creation of a cow with a sex ratio altering genetic change expected to lead to 3/4 of the cow's offspring being males. This type of sex ratio distortion results in gene drive and is also being considered to help control populations of invasive mammals.
Meet Cosmo the Frankenbull: Scientists genetically engineer a bull calf so that 75 per cent of its offspring will be male
13476J. Pinkstone, Daily Mail, 2020-07-24 20:58:14.
J. Pinkstone (2020). Daily Mail. A media report on the creation of a cow with a sex ratio altering genetic change expected to lead to 3/4 of the cow's offspring being males. This type of sex ratio distortion results in gene drive and is also being considered to help control populations of invasive mammals.
Meet the first genetically modified bull. Why did scientists change it
13473J. Kessler, Free News, 2020-07-23 20:52:51.
J. Kessler (2020). Free News. UC Davis scientists have successfully introduced a bovine embryo, or the bovine SRY gene, which is responsible for the development of the male. This is the first demonstration of targeted gene insertion for large DNA sequences through embryo-mediated genome editing in cattle. This type of sex ratio distortion results in gene drive and is also being considered to help control populations of invasive mammals.
Genome Editing 2020: Ethics and Human Rights in Germline Editing in Humans and Gene Drives in Mosquitoes
13198G. J. Annas, American Journal of Law and Medicine, 46:143-165. 2020-07-12 18:17:19.
G. J. Annas (2020). American Journal of Law and Medicine. doi: 10.1177/0098858820933492. I begin with a discussion of so far disastrously unsuccessful attempts to regulate germline editing in humans, including a summary of the first application of germline genome editing in humans and its aftermath. I then turn to a discussion of setting ethical standards for a genomic technology that has not yet been deployed in nature—gene drives. Finally, I end by suggesting that human rights can and should be directly applicable to defining the ethics of genomic research.
Small-Molecule Control of Super-Mendelian Inheritance in Gene Drives
12711V. López Del Amo, B. S. Leger, K. J. Cox, S. Gill, A. L. Bishop, G. D. Scanlon, J. A. Walker, V. M. Gantz and A. Choudhary, Cell Reports, 31:107841. 2020-06-30 20:53:02.
Summary Synthetic CRISPR-based gene-drive systems have tremendous potential in public health and agriculture, such as for fighting vector-borne diseases or suppressing crop pest populations. These elements can rapidly spread in a population by breaching the inheritance limit of 50% dictated by Mendel’s law of gene segregation, making them a promising tool for population engineering. However, current technologies lack control over their propagation capacity, and there are important concerns about potential unchecked spreading. Here, we describe a gene-drive system in Drosophila that generates an analog inheritance output that can be tightly and conditionally controlled to between 50% and 100%. This technology uses a modified SpCas9 that responds to a synthetic, orally available small molecule, fine-tuning the inheritance probability. This system opens a new avenue to feasibility studies for spatial and temporal control of gene drives using small molecules.
Development of zygotic and germline gene drives in mice
12531C. Pfitzner, J. N. Hughes, M. A. White, M. Scherer, S. G. Piltz and P. Q. Thomas, bioRxiv, 2020-06-21 15:15:40.
Here we investigated the efficiency of CRISPR/Cas9-based gene drives in Mus musculus by constructing "split drive" systems with Cas9 under the control of zygotic (CAG) or germline (Vasa) promoters.
CRISPR/Cas9 gene drive technology to control transmission of vector-borne parasitic infections
12386M. Nateghi Rostami, Parasite Immunology, preprint:e12762. 2020-06-04 18:10:02.
Gene drive is the process of copying of an endonuclease-containing cassette that leads to increased frequency of inheritance of the desired traits in a targeted population. CRISPR/Cas9 technology is advancing genetic manipulation of insects in the field of gene drive experiments. The CRISPR/Cas9 drive could be engineered for genetic manipulation of parasites and/or vectors for disease control. A number of promising CRISPR/Cas9-based gene drive strategies that interfere with parasite development or impairs the reproductive capability of the insect vector, have been proposed in the laboratory for blocking transmission of malaria and leishmaniasis. Still several technical and ethical challenges remain to be addressed, none appear insuperable in this field.
Can CRISPR gene drive work in pest and beneficial haplodiploid species?
12389J. Li, O. Aidlin Harari, A.-L. Doss, L. L. Walling, P. W. Atkinson, S. Morin and B. E. Tabashnik, Evolutionary Applications, 2020-06-03 18:14:33.
Gene drives based on CRISPR/Cas9 have the potential to reduce the enormous harm inflicted by crop pests and insect vectors of human disease, as well as to bolster valued species. In contrast with extensive empirical and theoretical studies in diploid organisms, little is known about CRISPR gene drive in haplodiploids, despite their immense global impacts as pollinators, pests, natural enemies of pests, and invasive species in native habitats. Here we analyze mathematical models demonstrating that, in principle, CRISPR homing gene drive can work in haplodiploids, as well as at sex-linked loci in diploids. However, relative to diploids, conditions favoring the spread of alleles deleterious to haplodiploid pests by CRISPR gene drive are narrower, the spread is slower, and resistance to the drive evolves faster. By contrast, the spread of alleles that impose little fitness cost or boost fitness was not greatly hindered in haplodiploids relative to diploids. Therefore, altering traits to minimize damage caused by harmful haplodiploids, such as interfering with transmission of plant pathogens, may be more likely to succeed than control efforts based on introducing traits that reduce pest fitness. Enhancing fitness of beneficial haplodiploids with CRISPR gene drive is also promising.
Simulation models from: Can CRISPER-mediated gene drive work in pest and beneficial haplodiploid species?
11968J. Li and B. Tabashnik, Dryad, 2020-05-26 18:17:03.
Gene drives based on CRISPR/Cas9 have the potential to reduce the enormous harm inflicted by crop pests and insect vectors of human disease, as well as to bolster valued species. In contrast with extensive empirical and theoretical studies in diploid organisms, little is known about CRISPR gene drive in haplodiploids, despite their immense global impacts as pollinators, pests, natural enemies of pests, and invasive species in native habitats. Here we analyze mathematical models demonstrating that, in principle, CRISPR homing gene drive can work in haplodiploids, as well as at sex-linked loci in diploids. However, relative to diploids, conditions favoring the spread of alleles deleterious to haplodiploid pests by CRISPR gene drive are narrower, the spread is slower, and resistance to the drive evolves faster. By contrast, the spread of alleles that impose little fitness cost or boost fitness was not greatly hindered in haplodiploids relative to diploids. Therefore, altering traits to minimize damage caused by harmful haplodiploids, such as interfering with transmission of plant pathogens, may be more likely to succeed than control efforts based on introducing traits that reduce pest fitness. Enhancing fitness of beneficial haplodiploids with CRISPR gene drive is also promising.
Le forçage génétique (gène drive) et ses applications
18210V. Courtier-Orgogozo, Bulletin de l'Académie Vétérinaire de France, 172:94-98. 2020-05-18 15:11:30.
Gene drive is a new genetic engineering technology that has been developed over the past five years and that allows genetic modifications to spread rapidly in natural populations. Potential applications are numerous, for public health issues, agriculture and conservation biology. This article presents the current developments in this biotechnology, as well as the issues and risks associated with it.
Malaria mosquitoes eliminated in lab by creating all male populations
11609H. Dunning, Imperial College London, 2020-05-11 16:07:00.
A team led by Imperial College London spread a genetic modification that distorts the sex ratio through a population of caged Anopheles gambiae mosquitoes using ‘gene drive’ technology.
A male-biased sex-distorter gene drive for the human malaria vector Anopheles gambiae
11463A. Simoni, A. M. Hammond, A. K. Beaghton, R. Galizi, C. Taxiarchi, K. Kyrou, D. Meacci, M. Gribble, G. Morselli, A. Burt, T. Nolan and A. Crisanti, Nature Biotechnology, 2020-05-11 15:15:55.
We report a male-biased sex-distorter gene drive (SDGD) in the human malaria vector Anopheles gambiae.
Converting endogenous genes of the malaria mosquito into simple non-autonomous gene drives for population replacement
11465A. Hoermann, S. Tapanelli, P. Capriotti, E. K. G. Masters, T. Habtewold, G. K. Christophides and N. Windbichler, bioRxiv, 2020-05-10 15:19:12.
Here we explore how minimal genetic modifications of endogenous mosquito genes can convert them directly into non-autonomous gene drives without disrupting their expression.
Gene drive outcomes not determined by genetic variation – A Podcast
11240Thomas Locke, Malaria Minute, 2020-04-29 17:23:49.
Gene drives are a system of genetic modification that use ‘molecular scissors’ to edit DNA sequences that self-perpetuate to ensure the rapid spread of mutation in a population. They offer new avenues for eradicating vector-borne diseases like malaria. They rely on the Cas9 system which works by recognising a specific 23-base pair stretch of DNA, assisted by a complementary RNA molecule which guides the enzyme and a donor DNA fragment that replaces the original DNA sequence. If there was genetic variation in the mosquito with respect to the RNA guide molecule used, however, it’s thought that such recognition would be less likely, meaning that the gene drive could fail. But a new study published this week suggests that this isn’t the case. Out of over 1,000 Anopheles mosquitoes analysed, around 90% had at least one target site for the Cas9 protein, suggesting that gene drives are still viable and not dependent on variation.
Can a population targeted by a CRISPR-based homing gene drive be rescued?
11269N. O. Rode, V. Courtier-Orgogozo and F. Débarre, bioRxiv, 2020.03.17.995829. 2020-03-20 19:12:23.
CRISPR-based homing gene drive is a genetic control technique aiming to modify or eradicate natural populations through the release of individuals carrying an engineered piece of DNA that can be inherited by all their progeny. Developing countermeasures is important to control the spread of gene drives, should they result in unanticipated damages. One proposed countermeasure is the introduction of individuals carrying a brake construct that targets and inactivates the drive allele but leaves the wild-type allele unaffected. Here we develop models to investigate the efficiency of such brakes. We consider a variable population size and use a combination of analytical and numerical methods to determine the conditions where a brake can prevent the extinction of a population targeted by an eradication drive. We find that a brake is not guaranteed to prevent eradication and that characteristics of both the brake and the drive affect the likelihood of recovering the wild-type population. In particular, brakes that restore fitness are more efficient than brakes that do not. Our model also suggests that threshold-dependent drives (drives that can spread only when introduced above a threshold) are more amenable to control with a brake than drives that can spread from an arbitrary low introduction frequency (threshold-independent drives). Based on our results, we provide practical recommendations and discuss safety issues.Article summary for Issue Highlights Homing gene drive is a new genetic control technology that aims to spread a genetically engineered DNA construct within natural populations even when it impairs fitness. In case of unanticipated damages, it has been proposed to stop homing gene drives by releasing individuals carrying a gene-drive brake; however, the efficiency of such brakes has been little studied. The authors develop a model to investigate the dynamics of a population targeted by a homing drive in absence or in presence of brake. The model provides insights for the design of more efficient brakes and safer gene drives.CRISPRClustered Regularly Interspaced Short Palindromic Repeats
Computational and experimental performance of CRISPR homing gene drive strategies with multiplexed gRNAs
7962S. E. Champer, S. Y. Oh, C. Liu, Z. Wen, A. G. Clark, P. W. Messer and J. Champer, Science Advances, 6:eaaz0525. 2020-03-16 14:22:56.
The rapid evolution of resistance alleles poses a major obstacle for genetic manipulation of populations with CRISPR homing gene drives. One proposed solution is using multiple guide RNAs (gRNAs), allowing a drive to function even if some resistant target sites are present. Here, we develop a model of homing mechanisms parameterized by experimental studies. Our model incorporates several factors affecting drives with multiple gRNAs, including timing of cleavage, reduction in homology-directed repair efficiency due to imperfect homology, Cas9 activity saturation, gRNA activity level variance, and incomplete homology-directed repair. We find that homing drives have an optimal number of gRNAs, usually between two and eight, depending on the specific drive type and performance parameters. These results contradict the notion that resistance rates can be reduced to arbitrarily low levels by gRNA multiplexing and highlight the need for combined approaches to counter resistance evolution in CRISPR homing drives.
Anti-CRISPR protein applications: natural brakes for CRISPR-Cas technologies
11201Marino, N. D., Pinilla-Redondo, R. , Csorgo, B., Bondy-Denomy, J., Nature Methods, 2020-03-16 13:52:42.
Clustered, regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes, a diverse family of prokaryotic adaptive immune systems, have emerged as a biotechnological tool and therapeutic. The discovery of protein inhibitors of CRISPR-Cas systems, called anti-CRISPR (Acr) proteins, enables the development of more controllable and precise CRISPR-Cas tools. Here we discuss applications of Acr proteins for post-translational control of CRISPR-Cas systems in prokaryotic and mammalian cells, organisms and ecosystems. This Review highlights recent discoveries and applications of anti-CRISPR (Acr) proteins that enable the regulation of CRISPR-Cas technology.
Population-level multiplexing: A promising strategy to manage the evolution of resistance against gene drives targeting a neutral locus
11212M. P. Edgington, T. Harvey-Samuel and L. Alphey, Evolutionary Applications, 10. 2020-03-06 14:52:40.
CRISPR-based gene drives bias inheritance in their favour by inducing double-stranded breaks (DSBs) at wild-type homologous loci and using the drive transgene as a repair template-converting drive heterozygotes into homozygotes. Recent studies have shown that alternate end-joining repair mechanisms produce cut-resistant alleles that rapidly induce drive failure. Multiplexing-simultaneously targeting multiple sites at the wild-type locus-is commonly assumed to overcome this issue since resistance would need to develop at all target sites for the system to fail. This may work for some population suppression drives targeting essential (e.g. viability or fertility) genes if careful design can ensure cut-resistant alleles themselves have low fitness. However, here, models are used to demonstrate that this approach will be ineffective when targeting neutral loci. We then go on to compare the performance of four alternative population-level multiplexing approaches with standard individual-level multiplexing. Two of these approaches have mechanisms preventing them from becoming linked, thus avoiding multiple simultaneous DSBs and giving a large improvement. Releasing multiple unlinked drives gives a modest improvement, while releasing multiple drives that may become linked over time produces a decrease in performance under the conditions tested here. Based on performance and technical feasibility, we then take one approach forward for further investigation, demonstrating its robustness to different performance parameters and its potential for controlling very large target populations.
Genome engineering in insects: focus on the CRISPR/Cas9 system
19405Hillary, V. Edwin Ceasar, Stanislaus Antony Ignacimuthu, S., Genome Engineering via CRISPR-Cas9 System, 2020-02-18 18:07:54.
Genome engineering is a precise tool used to alter the genome of desired organism. Zinc finger nuclease (ZFN), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR), and the CRISPR-associated RNA guided endonuclease Cas9 (CRISPR/Cas9) are the major genome engineering tools used in these days. CRISPR/Cas9 system has redeemed the precise genome engineering in different species including insects. This chapter covers the details on genome engineering studies reported in various insects including mosquitoes, butterflies, silkworm and fruit fly with a focus on CRISPR/Cas9 system. Many studies have been reported on application of ZFN, TALEN and CRISPR/Cas9 in insects. In recent years, many scientists have adopted CRISPR/Cas9 system for insect genome modification due to its affordability and quick designing of the constructs. We also discuss the details and applications of gene drive. Further studies with CRISPR/Cas9in insects will help researchers to find an effective strategy to combat the vector borne diseases spread by insects like mosquitoes.
Public Opinion Towards Gene Drive as a Pest Control Approach for Biodiversity Conservation and the Association of Underlying Worldviews
7348E. A. MacDonald, J. Balanovic, E. D. Edwards, W. Abrahamse, B. Frame, A. Greenaway, R. Kannemeyer, N. Kirk, F. Medvecky, T. L. Milfont, J. C. Russell and D. M. Tompkins, Environmental Communication-a Journal of Nature and Culture, 15:1-16. 2020-01-27 21:27:11.
Synthetic gene drive approaches are nascent technologies with potential applicability for pest control for conservation purposes. Responsible science mandates that society be engaged in a dialogue over new technology, particularly where there exist global ramifications as with gene drive. We hypothesize that public attitudes towards gene drive are not formed on scientific knowledge or demographics alone, but are heavily influenced by underlying worldviews, which encapsulate a broad and interactive system of attitudes, beliefs, and values. To test this, we conducted a national survey in New Zealand (n = 8199) and found that respondents clustered into four distinct segments with underlying worldviews, better able to explain attitudes toward gene drive than either the participants' scientific knowledge or other explanatory factors such demographics, political ideology or religiosity. We found that the use of gene drive for biodiversity conservation currently has moderate (32%) levels of support in New Zealand but that varied substantially across the four segments. Should gene drive become a technically viable approach for pest control, understanding the worldviews that shape public decision-making can guide a more empathetic engagement process and empower society to participate in informed decision-making about if and how gene drive should be used for conservation purposes.
Development of a confinable gene drive system in the human disease vector Aedes aegypti
6697M. Li, T. Yang, N. P. Kandul, M. Bui, S. Gamez, R. Raban, J. Bennett, H. M. Sánchez C, G. C. Lanzaro, H. Schmidt, Y. Lee, J. M. Marshall and O. S. Akbari, eLife, 9:e51701. 2020-01-16 18:07:34.
Aedes aegypti is the principal mosquito vector for many arboviruses that increasingly infect millions of people every year. With an escalating burden of infections and the relative failure of traditional control methods, the development of innovative control measures has become of paramount importance. The use of gene drives has sparked significant enthusiasm for genetic control of mosquitoes; however, no such system has been developed in Ae. aegypti. To fill this void, here we develop several CRISPR-based split gene drives for use in this vector. With cleavage rates up to 100% and transmission rates as high as 94%, mathematical models predict that these systems could spread anti-pathogen effector genes into wild populations in a safe, confinable and reversible manner appropriate for field trials and effective for controlling disease. These findings could expedite the development of effector-linked gene drives that could safely control wild populations of Ae. aegypti to combat local pathogen transmission.
The kill-switch for CRISPR that could make gene-editing safer
6647E. Dolgin, Nature, 577:308-310. 2020-01-15 20:32:43.
How anti-CRISPR proteins and other molecules could bolster biosecurity and improve medical treatments.
These are the 5 most dystopian technologies of 2020 and beyond
6188M. Sullivan, FastCompany, 2019-12-28 19:13:46.
Tech is always both good and bad. But we live in a time when everything gets weaponized—ideas, images, ancient texts, biases, and even people. And technology provides the tools to do it easier, faster, and with less resources.
Design and analysis of CRISPR-based underdominance toxin-antidote gene drives
5626Champer, J., S. E. Champer, I. Kim, A. G. Clark and P. W. Messer, bioRxiv, 861435:861435. 2019-12-17 16:38:34.
CRISPR gene drive systems offer a mechanism for transmitting a desirable transgene throughout a population for purposes ranging from vector-borne disease control to invasive species suppression. In this simulation study, we model and assess the performance of several CRISPR-based underdominance gene drive constructs employing toxin-antidote principles. These drives disrupt the wild-type version of an essential gene using a CRISPR nuclease (the toxin) while simultaneously carrying a recoded version of the gene (the antidote). Drives of this nature allow for releases that could be potentially confined to a desired geographic location. This is because such drives have a nonzero invasion threshold frequency, referring to the critical frequency required for the drive to spread through the population. We model drives which target essential genes that are either haplosufficient or haplolethal, using nuclease promoters with expression restricted to the germline, promoters that additionally result in cleavage activity in the early embryo from maternal deposition, and promoters that have ubiquitous somatic expression. We also study several possible drive architectures, considering both “same-site” and “distant-site” systems, as well as several reciprocally targeting drives. Together, these drive variants provide a wide range of invasion threshold frequencies and options for both population modification and suppression. Our results suggest that CRISPR toxin-antidote underdominance drive systems could allow for the design of highly flexible and potentially confinable gene drive strategies.
Gene drive and resilience through renewal with next generation Cleave and Rescue selfish genetic elements
5602Oberhofer, G., T. Ivy and B. A. Hay, bioRxiv, 2019:2019.2012.2013.876169. 2019-12-17 16:01:29.
Gene drive-based strategies for modifying populations face the problem that genes encoding cargo and the drive mechanism are subject to separation, mutational inactivation, and loss of efficacy. Resilience, an ability to respond to these eventualities in ways that restore population modification with functional genes is needed for long-term success. Here we show that resilience can be achieved through cycles of population modification with Cleave and Rescue (ClvR) selfish genetic elements. ClvR comprises a DNA sequence-modifying enzyme such as Cas9/gRNAs that disrupts endogenous versions of an essential gene, and a recoded version of the essential gene resistant to cleavage. ClvR spreads by creating conditions in which those lacking ClvR die because they lack functional versions of the essential gene. Cycles of modification can in principal be carried out if two ClvR elements targeting different essential genes are located at the same genomic position, and one of them, ClvRn+1, carries a Rescue transgene from an earlier element, ClvRn. ClvRn+1 should spread within a population of ClvRn, while also bringing about a decrease in its frequency. To test this hypothesis we first show that multiple ClvRs, each targeting a different essential gene, function when located at a common chromosomal position in Drosophila. We then show that when several of these also carry the Rescue from a different ClvR, they spread to transgene fixation in populations fixed for the latter, and at its expense. Therefore, genetic modifications of populations can be overwritten with new content, providing an ongoing point of control.
Development of genetic control strategies for insect pests using CRISPR/Cas9 Développement de méthodes de lutte génétique contre de l’insecte nuisible basé sur le system CRISPR/Cas9
18505E. Green, Université de Strasbourg, 2019-12-17 14:20:21.
nsect pest control remains an important economic, environmental, and public health challenge. CRISPR/Cas9 gene drive (GD) is a novel genetic control strategy. GDs are genetic systems that can rapidly invade a population. This manuscript presents my efforts to develop gene drives in two important pest species, Anopheles gambiae, a major vector of malaria, and Drosophila suzukii, a global crop pest. The goals of this project were to develop a suppression gene drive in D. suzukii, to reduce population size, and a modification drive in An. gambiae, to reduce malaria transmission. While I was unable to produce a functional gene drive in D. suzukii, the efforts and protocols presented here can serve as a baseline for future work in this economically important crop pest. In An. gambiae, I successfully characterized two transgenic lines, one of which significantly blocks malaria transmission to a rodent model. Finally, I present my efforts to engineer a new modification gene drive strategy, indirect gene drive.
What is genome editing?
14784NHGRI, NHGRI, 2019-08-15 17:41:15.
Genome editing is a method that lets scientists change the DNA of many organisms, including plants, bacteria, and animals. Editing DNA can lead to changes in physical traits, like eye color, and disease risk. Scientists use different technologies to do this.
Improving plant-resistance to insect-pests and pathogens: The new opportunities through targeted genome editing
6176D. S. Bisht, V. Bhatia and R. Bhattacharya, Seminars in Cell & Developmental Biology, 96:65-76. 2019-05-08 18:53:44.
The advantages of high input agriculture are fading away due to degenerating soil health and adverse effects of climate change. Safeguarding crop yields in the changing environment and dynamics of pest and pathogens, has posed new challenges to global agriculture. Thus, integration of new technologies in crop improvement has been imperative for achieving the breeding objectives in faster ways. Recently, enormous potential of genome editing through engineered nucleases has been demonstrated in plants. Continuous refinements of the genome editing tools have increased depth and breadth of their applications. So far, genome editing has been demonstrated in more than fifty plant species. These include model species like Arabidopsis, as well as important crops like rice, wheat, maize etc. Particularly, CRISPR/Cas9 based two component genome editing system has been facile with wider applicability. Potential of genome editing has unfurled enormous possibilities for engineering diverse agronomic traits including durable resistance against insect-pests and pathogens. Novel propositions of developing insect and pathogen resistant crops by genome editing include altering the effector-target interaction, knocking out of host-susceptibility genes, engineering synthetic immune receptor eliciting broad spectrum resistance, uncoupling of antagonistic action of defense hormones etc. Alternatively, modification of insect genomes has been used either to create gene drive or to counteract resistance to various insecticides. The distinct advantage of genome editing system is that it can knock out specific target region in the genome without leaving the unwanted vector backbone. In this article, we have reviewed the novel opportunities offered by the genome editing technologies for developing insect and pathogen resistant crop-types, their future prospects and anticipated challenges.
Genetic pest management technologies to control invasive rodents
11576D. Kanavy and D. Threadgill, Island invasives: scaling up to meet the challenge, 2019-03-05 15:20:05.
Many strategies exist to manage invasive pests on islands, ranging from poison to trapping, with varying degrees of success. Genetic technologies are increasingly being applied to insect pests, but so far, not to vertebrates. We are implementing a genetic strategy to eradicate invasive mouse populations as another tool for pest control. Mus musculus, the common house mouse, is one of the most widespread invasive species. Mice threaten human health, agriculture, and biodiversity on many islands, particularly seabirds. Seabirds are endangered indirectly through competition for resources or predators being attracted by the mice or directly with mice attacking chicks and eggs. Rodenticides are the most common method of eradicating mice, but their use leads to poisoning of non-target species and has limited efficacy against mice. An approach that could eliminate non-target species impact would be to engineer daughterless mice linked to a gene drive system for self-sustained propagation. For this project, we have investigated exploiting a naturally occurring gene drive, the t-complex. Using the t w2 haplotype of the t-complex, we observed the t w2 haplotype being transmitted to offspring with a transmission distortion ratio of 95.3%. The daughterless phenotype is being accomplished by inserting the Sry gene (male sex-determining gene) into an autosome containing the tw2 haplotype via CRISPR/Cas9 gene editing. The presence of Sry will induce testis formation, regardless of the sex chromosomes naturally inherited. When Sry is inserted into the t-complex, the desired gene will spread through the population, eliminating female offspring. This model system will support studies to evaluate the effectiveness of crashing an invasive population without adversely affecting other
Trialling gene drives to control invasive species: what, where and how?
11573T. Harvey-Samuel, K. J. Campbell, M. Edgington and L. Alphey, Island invasives: scaling up to meet the challenge, 2019-03-05 15:16:48.
The control of invasive species would be enhanced through the addition of novel, more effective and sustainable pest management methods. One control option yet to be trialled in the field is to deploy transgene-based ‘Gene Drives’: technologies which force the inheritance of a genetic construct through the gene pool of a wild population, suppressing it or replacing it with a less harmful form. There is considerable interest in applying gene drives to currently intractable invasives across a broad taxonomic range. However, not all species will make efficient or safe targets for these technologies. Additionally, the safety and efficacy of these systems will vary according to where they are deployed, the specific molecular design chosen, and how these factors interact with the ecology of the target pest. Given the transformative but also controversial nature of gene drives, it is imperative that their first field trials are able to successfully demonstrate that they can be used safely and efficiently. Here, we discuss how to maximise the probability of this outcome through considering three important questions: What types of invasive species should we use to trial gene drives? Where should we be trialling them? and How should these trials be conducted? In particular, we focus on the ecological, genetic and geographic features of small, isolated islands which make them ideal locations for these initial trials. A case study of an island invasive that is deemed highly appropriate for gene drive intervention, and for which gene drive development is currently underway (Mus musculus), is used to further explore these concepts
Towards a genetic approach to invasive rodent eradications: assessing reproductive competitiveness between wild and laboratory mice
11570M. Serr, N. Heard and J. Godwin, Island invasives: scaling up to meet the challenge, 2019-03-05 15:10:12.
House mice are significant invasive pests, particularly on islands without native mammalian predators. As part of a multi-institutional project aimed at suppressing invasive mouse populations on islands, we aim to create heavily male-biased sex ratios with the goal of causing the populations to crash. Effective implementation of this approach will depend on engineered F1 wild-lab males being effective secondary invaders that can mate successfully. As a first step in assessing this possibility, we are characterising genetic and behavioural differences between Mus musculus strains in terms of mating and fecundity using wild house mice derived from an invasive population on the Farallon Islands (MmF), a laboratory strain C57BL/6/129 (tw2), and F1 wild-lab off spring. Mice with the ‘t allele’ (tw2) have a naturally occurring gene drive system. To assess fertility in F1 wild-lab crosses, tw2 males were paired with wild-derived females from the Farallon Islands (MmF). Results of these matings indicate litter sizes are comparable but that weaned pup and adult wild-lab mice are heavier in mass. Next, we initiated tests of male competitiveness using larger (3 m2 ) enclosures with enrichment. We introduced both an MmF and a tw2-bearing male to two MmF females to assess mating outcomes. Preliminary results of these experiments show none of the offspring carried the t-allele. However, performing the same experiment with F1 wildlab males instead of a full lab background resulted in 70% of off spring carrying the t w2 allele. This indicates that F1 wildlab males may be able to successfully compete and secondarily invade. It will be important in subsequent experiments to determine what characteristics contribute to secondary invasion success. More generally, a better understanding of characteristics contributing to overall success in increasingly complex and naturalistic environments will be critical in determining the potential of a gene drive-based eradication approach for invasive mice on islands
A potential new tool for the toolbox: assessing gene drives for eradicating invasive rodent populations
11550K. J. Campbell, J. R. Saah, P. R. Brown, J. Godwin, F. Gould, G. R. Howald, A. Piaggio, P. Thomas, D. M. Tompkins, D. Threadgill, J. Delborne, D. Kanavy, T. Kuiken, H. Packard, M. Serr and A. Shiels, Island invasives: scaling up to meet the challenge, 2019-03-05 14:59:34.
Invasive rodents have significant negative impacts on island biodiversity. All but the smallest of rodent eradications currently rely on island-wide rodenticide applications. Although signifi cant advances have been made in mitigating unintended impacts, rodent eradication on inhabited islands remains extremely challenging. Current tools restrict eradication eff orts to fewer than 15% of islands with critically endangered or endangered species threatened by invasive rodents. The Genetic Biocontrol of Invasive Rodents partnership is an interdisciplinary collaboration to develop and evaluate gene drive technology for eradicating invasive rodent populations on islands. Technological approaches currently being investigated include the production of multiple strains of Mus musculus with a modifi ed form of the native t-complex, or a CRISPR gene drive, carrying genes or mechanisms that determine sex. These systems have the potential to skew the sex ratio of off spring to approach 100% single-sex, which could result in population collapse. One goal proposed is to test the ability of constructs to spread and increase in frequency in M. musculus populations in biosecure, captive settings and undertake modelling to inform development and potential deployment of these systems. Structured ecologically-based risk assessments are proposed, along with social and cultural engagement to assess the acceptability of releasing a gene drive system. Work will be guided by an external ethics advisory board. Partners are from three countries with significant regulatory capacity (USA, Australia, New Zealand). Thus, we will seek data sharing agreements so that results from experiments may be used within all three countries and treat regulatory requirements as a minimum. Species-specific, scalable, and socially acceptable new eradication tools could produce substantial biodiversity benefits not possible with current technologies. Gene drive innovation may provide such a tool for invasive species management and be potentially transformative and worthy of exploring in an inclusive, responsible, and ethical manner.
Modulating CRISPR gene drive activity through nucleocytoplasmic localization of Cas9 in S. cerevisiae
18839M. E. Goeckel, E. M. Basgall, I. C. Lewis, S. C. Goetting, Y. Yan, M. Halloran and G. C. Finnigan, Fungal Biology Biotechnology, 6:2. 2019-02-16 13:04:41.
In this study, we use artificial gene drives in budding yeast to explore mechanisms to modulate nuclease activity of Cas9 through its nucleocytoplasmic localization. We examine non-native nuclear localization sequences (both NLS and NES) on Cas9 fusion proteins in vivo through fluorescence microscopy and genomic editing. Our results demonstrate that mutational substitutions to nuclear signals and combinatorial fusions can both modulate the level of gene drive activity within a population of cells.
CRISPR gene drive efficiency and resistance rate is highly heritable with no common genetic loci of large effect
3901Champer, JW, Z. X.; Luthra, A.; Reeves, R.; Chung, J.; Liu, C.; Lee, Y. L.; Liu, J. X.; Yang, E.; Messer, P. W.; Clark, A. G., Genetics, 212:333-341. 2019-01-19 00:00:00.
Gene drives could allow for control of vector-borne diseases by directly suppressing vector populations or spreading genetic payloads designed to reduce pathogen transmission. Clustered regularly interspaced short palindromic repeat (CRISPR) homing gene drives work by cleaving wild-type alleles, which are then converted to drive alleles by homology-directed repair, increasing the frequency of the drive in a population over time. However, resistance alleles can form when end-joining repair takes place in lieu of homology-directed repair. Such alleles cannot be converted to drive alleles, which would eventually halt the spread of a drive through a population. To investigate the effects of natural genetic variation on resistance formation, we developed a CRISPR homing gene drive in Drosophila melanogaster and crossed it into the genetically diverse Drosophila Genetic Reference Panel (DGRP) lines, measuring several performance parameters. Most strikingly, resistance allele formation postfertilization in the early embryo ranged from 7 to 79% among lines and averaged 42 +/- 18%. We performed a genome-wide association study using our results in the DGRP lines, and found that the resistance and conversion rates were not explained by common alleles of large effect, but instead there were several genetic polymorphisms showing weak association. RNA interference knockdown of several genes containing these polymorphisms confirmed their effect, but the small effect sizes imply that their manipulation would likely yield only modest improvements to the efficacy of gene drives.
A Multiple Gene Drive System
4719Ferdinand Nanfack Minkeu, IGTRCN, 2019-01-14 00:00:00.
Yan & Finnigan, (2018) recently published a paper in Scientific Reports describing an artificial multi-locus gene drive system by using a single Cas9 and three guide RNA (gRNA) in the budding yeast Saccharomyces cerevisiae. Nuclease-based gene drives do not follow the typical Mendel patterns of inheritance and lead quickly to the spread of gene drive elements through a given population.
Super-Mendelian inheritance mediated by CRISPR-Cas9 in the female mouse germline
3914Grunwald, HAG, V. M.; Poplawski, G.; Xu, X. R. S.; Bier, E.; Cooper, K. L., Nature, 566:105-109. 2019-01-12 00:00:00.
A gene drive biases the transmission of one of the two copies of a gene such that it is inherited more frequently than by random segregation. Highly efficient gene drive systems have recently been developed in insects, which leverage the sequence-targeted DNA cleavage activity of CRISPR-Cas9 and endogenous homology-directed repair mechanisms to convert heterozygous genotypes to homozygosity(1-4). If implemented in laboratory rodents, similar systems would enable the rapid assembly of currently impractical genotypes that involve multiple homozygous genes (for example, to model multigenic human diseases). To our knowledge, however, such a system has not yet been demonstrated in mammals. Here we use an active genetic element that encodes a guide RNA, which is embedded in the mouse tyrosinase (Tyr) gene, to evaluate whether targeted gene conversion can occur when CRISPR-Cas9 is active in the early embryo or in the developing germline. Although Cas9 efficiently induces double-stranded DNA breaks in the early embryo and male germline, these breaks are not corrected by homology-directed repair. By contrast, Cas9 expression limited to the female germline induces double-stranded breaks that are corrected by homology-directed repair, which copies the active genetic element from the donor to the receiver chromosome and increases its rate of inheritance in the next generation. These results demonstrate the feasibility of CRISPR-Cas9-mediated systems that bias inheritance of desired alleles in mice and that have the potential to transform the use of rodent models in basic and biomedical research.
CRISPR-Cas9. The greatest advancement in genetic edition techniques requires an ethical reflection
3913Gomez-Tatay, LA, J., Cuadernos De Bioetica, 30:171-185. 2019-01-11 00:00:00.
The adaptation of the CRISPR system as a genetic editing tool has led to a revolution in many fields of application, as this technique is considerably faster, easier to perform and more efficient than predecessor techniques. However, some of these applications raise objective ethical issues that must be addressed. In this paper we discuss, based on the most recent data, the different issues related to CRISPR applications on the germ line, its introduction in clinical trials, the genetic edition of animals and plants for human consumption and the novel gene drive.
Gene driving the farm: who decides, who owns, and who benefits?
3907Montenegro de Wit, M, Agroecology and Sustainable Food Systems, 43:1054-1074. 2019-01-05 00:00:00.
This commentary essay explores the social and ecological implications of gene-driving agriculture.
Multiplexing gRNAs to Hedge Against Resistance to Gene Drive
4720Travis Van Warmerdam, IGTRCN, 2018-11-25 00:00:00.
Recently, Oberhofer et al (2018) published a paper examining the mechanisms of homing endonuclease gene drives in The Proceedings of the National Academy of Sciences. They used a novel nuclease-encoding cassette containing four multiplexed gRNAs targeting genes required for viability or female fertility in Drosophila melanogaster.
Cas9 Gene Drive, Sex-Conversion and Evolved Resistance
4721Anna Buchman, IGTRCN, 2018-09-24 00:00:00.
In a recent manuscript, KaramiNejadRanjbar et al. demonstrate the development of a proof of principle Cas9-based suppression gene drive in D. melanogaster that can be applied to pest insects, and discuss the implications of resistance allele formation for practical use of such a drive system.
Pest demography critically determines the viability of synthetic gene drives for population control
11505K. E. Wilkins, T. A. A. Prowse, P. Cassey, P. Q. Thomas and J. V. Ross, Mathematical Biosciences, 305:160-169. 2018-09-13 20:48:39.
Synthetic gene drives offer a novel solution for the control of invasive alien species. CRISPR-based gene drives can positively bias their own inheritance, and comprise a DNA sequence that is replicated by homologous recombination. Since gene drives can be positioned to silence fertility or developmental genes, they could be used for population suppression. However, the production of resistant alleles following self-replication errors threatens the technology's viability for pest eradication in real-world applications. Further, a robust assessment of how pest demography impacts the expected progression of gene drives through populations is currently lacking. We used a deterministic, two-sex, birth-death model to investigate how demographic assumptions affect the efficiency of suppression drives for controlling invasive rodents on islands, for two different gene-drive strategies. We show that mass-action reproduction results in overly optimistic eradication outcomes when compared to the more realistic assumption of polygynous breeding. When polygyny was assumed, both gene-strategies failed due to the evolution of resistance unless a reproductive Allee effect (reduced reproductive rates at low population density) was also included; although model outcomes were highly sensitive to the strength of this effect. Increasing the size of the initial gene-drive introduction (up to 10% of carrying capacity) had little impact on population outcomes. Understanding the demography of a population targeted for eradication is critical before the viability of gene-drive suppression can be adequately assessed.
A sustainable synthetic biology approach for the control of the invasive golden mussel (Limnoperna fortunei)
19204M. F. Rebelo, L. F. Afonso, J. A. Americo, L. da Silva, J. L. B. Neto, F. Dondero and Q. Zhang, PeerJ Preprints, 6:e27164v3. 2018-09-12 14:52:07.
The recent development of the CRISPR-Cas9-based gene drive has created the conditions to seriously consider this technology to solve one of the major environmental challenges in biodiversity conservation i.e. the control of invasive species. There is no efficient control method for golden mussel infestation available so far. Here we discuss the technical and economic feasibility of using a synthetic biology based approach to fight and control the invasive mussel Limnoperna fortunei in South American rivers and reservoirs.
CRISPR Explained
14780Mayo Clinic, 2018-07-24 17:25:24.
A short video that simply explains what CRISPR is and how it is used for gene editing. Simple language and highly accessible.
What is CRISPR?
14778A. Vidyasagar, LiveScience, 2018-04-21 17:17:15.
CRISPR technology is a simple yet powerful tool for editing genomes. It allows researchers to easily alter DNA sequences and modify gene function. Its many potential applications include correcting genetic defects, treating and preventing the spread of diseases and improving crops. However, its promise also raises ethical concerns.
Current CRISPR gene drive systems are likely to be highly invasive in wild populations
4002Noble, CA, Ben; Church, George M.; Esvelt, Kevin M.; Nowak, Martin A., eLife, 7:e33423. 2018-01-20 00:00:00.
Recent reports have suggested that self-propagating CRISPR-based gene drive systems are unlikely to efficiently invade wild populations due to drive-resistant alleles that prevent cutting. Here we develop mathematical models based on existing empirical data to explicitly test this assumption for population alteration drives. Our models show that although resistance prevents spread to fixation in large populations, even the least effective drive systems reported to date are likely to be highly invasive. Releasing a small number of organisms will often cause invasion of the local population, followed by invasion of additional populations connected by very low rates of gene flow. Hence, initiating contained field trials as tentatively endorsed by the National Academies report on gene drive could potentially result in unintended spread to additional populations. Our mathematical results suggest that self-propagating gene drive is best suited to applications such as malaria prevention that seek to affect all wild populations of the target species.
Gene drive systems: Do they have a place in agricultural weed management?
4000Neve, P, Pest Management Science, 74:2672-2679. 2018-01-18 00:00:00.
There is a pressing need for novel control techniques in agricultural weed management. Direct genetic control of agricultural pests encompasses a range of techniques to introduce and spread novel, fitness-reducing genetic modifications through pest populations. Recently, the development of CRISPR-Cas9 gene editing has brought these approaches into sharper focus. Proof of concept for CRISPR-Cas9 based gene drives has been demonstrated for control of disease-vectoring insects. This article considers whether and how gene drives may be applied in agricultural weed management, focusing on CRISPR-Cas9 based systems. Population suppression drives might be employed to introduce and proliferate deleterious mutations that directly impact fitness and weediness, whereas population sensitizing drives would seek to edit weed genomes so that populations are rendered more sensitive to subsequent management interventions. Technical challenges relating to plant transformation and gene editing in planta are considered, and the implementation of gene drives for timely and sustainable weed management is reviewed in the light of weed population biology. The technical, biological, practical and regulatory challenges remain significant. Modelling-based studies can inform how and if gene drives could be employed in weed populations. These studies are an essential first step towards determining the utility of gene drives for weed management. This article is protected by copyright. All rights reserved.
Identifying and detecting potentially adverse ecological outcomes associated with the release of gene-drive modified organisms
3980Hayes, KRH, G. R.; Dana, G. V.; Foster, S. D.; Ford, J. H.; Thresher, R.; Ickowicz, A.; Peel, D.; Tizard, M.; De Barro, P.; Strive, T.; Dambacher, J. M., Journal of Responsible Innovation, 5:S139-S158. 2018-01-18 00:00:00.
Synthetic gene drives could provide new solutions to a range of old problems such as controlling vector-borne diseases, agricultural pests and invasive species. In this paper, we outline methods to identify hazards and detect potentially adverse ecological outcomes at the individual (genotype, phenotype), population, community and ecosystem level, when progressing Gene Drive Modified Organisms through a phased test and release pathway. We discuss the strengths and weaknesses of checklists and structured hazard analysis techniques, identify methods to help meet some of the challenges of detecting adverse ecological outcomes in experiments and confined field trials, and discuss ways to improve the efficiency and statistical rigour of post-release monitoring strategies.
Identifying knowledge gaps for gene drive research to control invasive animal species: The next CRISPR step
3998Moro, DB, Margaret; Kennedy, Malcolm; Campbell, Susan; Tizard, Mark, Global Ecology and Conservation, 13:e00363. 2018-01-16 00:00:00.
Invasive animals have been linked to the extinctions of native wildlife, and to significant agricultural financial losses or impacts. Current approaches to control invasive species require ongoing resources and management over large geographic scales, and often result in the short-term suppression of populations. New and innovative approaches are warranted. Recently, the RNA guided gene drive system based on CRISPR/Cas9 is being proposed as a potential gene editing tool that could be used by wildlife managers as a nonlethal addition or alternative to help reduce pest animal populations. While regulatory control and social acceptance are crucial issues that must be addressed, there is an opportunity now to identify the knowledge and research gaps that exist for some important invasive species. Here we systematically determine the knowledge gaps for pest species for which gene drives could potentially be applied. We apply a conceptual ecological risk framework within the gene drive context within an Australian environment to identify key requirements for undertaking work on seven exemplar invasive species in Australia. This framework allows an evaluation of the potential research on an invasive species of interest and within a gene drive and risk context. We consider the currently available biological, genetic and ecological information for the house mouse, European red fox, feral cat, European rabbit, cane toad, black rat and European starling to evaluate knowledge gaps and identify candidate species for future research. We discuss these findings in the context of future thematic areas of research worth pursuing in preparation for a more formal assessment of the use of gene drives as a novel strategy for the control of these and other invasive species.
Economic issues to consider for gene drives
3997Mitchell, PDB, Z.; McRoberts, N., Journal of Responsible Innovation, 5:S180-S202. 2018-01-15 00:00:00.
We examine four economic issues regarding gene drive applications made possible by gene editing technologies. First, whether gene drives are self-sustaining or self-limiting will largely determine which types of organizations have incentives to develop and deploy gene drives and greatly influence their governance and regulation. Social factors will also play key roles, particularly public perceptions, with these perceptions co-determined with regulation and governance. Second, gene drive applications will generate unintended negative social impacts that will partially offset benefits. Third, economic surplus, the traditional measure of economic benefits, incompletely captures the welfare impacts of gene drive applications. Fourth, gene drives imply dynamic nonlinearities that make identifying economic equilibria and general policy recommendations challenging. The potentially substantial benefits, coupled with the technical, social, and economic uncertainties surrounding gene drives, suggest that a responsible course of action is to move forward while maintaining regulatory flexibility and conducting research to resolve key uncertainties.
Gene drive inhibition by the anti-CRISPR proteins AcrIIA2 and AcrIIA4 in Saccharomyces cerevisiae
3957Basgall, EMG, S. C.; Goeckel, M. E.; Giersch, R. M.; Roggenkamp, E.; Schrock, M. N.; Halloran, M.; Finnigan, G. C., Microbiology-Sgm, 164:464-474. 2018-01-15 00:00:00.
Given the widespread use and application of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas gene editing system across many fields, a major focus has been the development, engineering and discovery of molecular means to precisely control and regulate the enzymatic function of the Cas9 nuclease. To date, a variety of Cas9 variants and fusion assemblies have been proposed to provide temporally inducible and spatially controlled editing functions. The discovery of a new class of 'anti-CRISPR' proteins, evolved from bacteriophage in response to the prokaryotic nuclease-based immune system, provides a new platform for control over genomic editing. One Cas9-based application of interest to the field of population control is that of the 'gene drive'. Here, we demonstrate use of the AcrIIA2 and AcrIIA4 proteins to inhibit active gene drive systems in budding yeast. Furthermore, an unbiased mutational scan reveals that titration of Cas9 inhibition may be possible by modification of the anti-CRISPR primary sequence.
CRISPR/Cas9 -mediated gene knockout of Anopheles gambiae FREP1 suppresses malaria parasite infection
3973Dong, YS, Maria L.; Marois, Eric; Dimopoulos, George, PLOS Pathogens, 14:e1006898. 2018-01-11 00:00:00.
The causative agent of malaria, Plasmodium, has to complete a complex infection cycle in the Anopheles gambiae mosquito vector in order to reach the salivary gland from where it can be transmitted to a human host. The parasite’s development in the mosquito relies on numerous host factors (agonists), and their inhibition or inactivation can thereby result in suppression of infection and consequently malaria transmission. The recently developed CRISPR/Cas9-based genome editing tools for Anopheles mosquitoes provide new and promising opportunities to delete (inactivate) Plasmodium agonists to better understand their function and for blocking malaria transmission. Here we have established a modified CRISPR/Cas9 genome editing technique for malaria vector A. gambiae mosquitoes. Through this approach we have inactivated the fibrinogen-related protein 1 (FREP1) gene, via CRISPR/Cas9 genome editing, and the impact of this manipulation on the mosquito’s susceptibility to Plasmodium and on mosquito fitness. FREP1 knockout mutants showed a profound suppression of infection with both human and rodent malaria parasites, while it also resulted in fitness costs: a significantly lower blood-feeding propensity, fecundity and egg hatching rate, and a retarded larval development and pupation time, and reduced longevity after a blood meal.
Genome editing: scientific opportunities, public interests and policy options in the European Union
16075EASAC, European Academies Science Advisory Council, 2017-03-01 14:41:21.
In many of the areas in which EASAC, the European Academies’ Science Advisory Council, works, where a large and solid body of knowledge is needed to inform the action of our societies, it is important to recognise that there is an intimate mix of science and values involved in discussion. Such discussions are most fruitful when both knowledge and values are well identified. This report presents a broad synthesis of genome editing, one of the newer aspects of the biosciences. It is our hope that presenting clearly the science involved – the duty of academies – will serve the ongoing discussions within society that the report recommends be vigorously pursued.
Gene drives do not always increase in frequency: from genetic models to risk assessment
4036de Jong, TJ, Journal Fur Verbraucherschutz Und Lebensmittelsicherheit-Journal of Consumer Protection and Food Safety, 12:299-307. 2017-01-14 00:00:00.
Homing genes encode endonucleases that make a double stranded break in the DNA, destroying a target site on the homologous chromosome. When the cell repairs the break the homing allele is copied, converting a heterozygote into a homozygote. This results in gene drive (GD), an overrepresentation of the homing allele in the next generation. GD may propel CRISPR-Cas9 genes and new genes physically coupled to the GD through natural populations. I revisit the population genetic models of GD with the aim of making these models more understandable to non-specialists. What can we learn about risk evaluation from the models? A GD with no or a small effect on fitness (viability) always spreads in the population and goes to fixation. That is provided that no resistance mechanism evolves, for instance due to a mutation in the target site. However, when GDs have a large negative effect on fitness, their spread depends on a threshold or they may not spread at all. The chance of GDs increasing until fixation is much higher in systems with meiotic drive than in systems with embryo conversion. The presence or absence of a meiotic promoter is therefore relevant to take into account in the environmental risk assessment.
The promise and peril of CRISPR gene drives
4076Zentner, GEW, Michael J. C., Bioessays, 39:1-9. 2017-01-14 00:00:00.
Gene drives are selfish genetic elements that use a variety of mechanisms to ensure they are transmitted to subsequent generations at greater than expected frequencies. Synthetic gene drives based on the clustered regularly interspersed palindromic repeats (CRISPR) genome editing system have been proposed as a way to alter the genetic characteristics of natural populations of organisms relevant to the goals of public health, conservation, and agriculture. Here, we review the principles and potential applications of CRISPR drives, as well as means proposed to prevent their uncontrolled spread. We also focus on recent work suggesting that factors such as natural genetic variation and inbreeding may represent substantial impediments to the propagation of CRISPR drives.
The End of the GMO? Genome Editing, Gene Drives and New Frontiers of Plant Technology
15368K. L. Hefferon and R. J. Herring, Review of Agrarian Studies, 7. 2017-01-01 21:40:33.
mprovements to agriculture will constitute one of the world’s greatest challenges in the coming century. Political and social controversies, as well as complications of plant breeding, intellectual property, and regulation, have compromised the promised impact of genetically engineered – typically transgenic – crops designated as “GMOs.” Genome editing is a new suite of molecular tools for assisting biologists identify genes that control agronomic traits such as drought tolerance and pest resistance, as well as to elucidate how expression of these genes is intertwined within the functional framework of the cell. This technology has recently gained momentum for its ability to accelerate the crop breeding process in an unprecedented fashion and expand the range of crop varieties with improved precision and lower costs. This review explains the basic concepts and provides examples of how genome editing could help address the United Nation’s Sustainable Development Goals with respect to food, agriculture, and medicine. It concludes with a discussion of the potential social impact of genome editing and gene drive. These effects are contingent on the resolution of novel ethical and regulatory challenges that add new layers of complexity to societal questions of appropriate technology, in agriculture and beyond. We expect these questions to replace the irresolvable GMO debate.
Prospects and challenges of CRISPR/Cas genome editing for the study and control of neglected vector-borne nematode diseases
6017M. Zamanian and E. C. Andersen, The FEBS Journal, 283:3204-3221. 2016-06-14 20:22:18.
Neglected tropical diseases caused by parasitic nematodes inflict an immense health and socioeconomic burden throughout much of the developing world. Current estimates indicate that more than two billion people are infected with nematodes, resulting in the loss of 14 million disability-adjusted life years per annum. Although these parasites cause significant mortality, they primarily cause chronic morbidity through a wide range of severe clinical ailments. Treatment options for nematode infections are restricted to a small number of anthelmintic drugs, and the rapid expansion of anthelmintic mass drug administration raises concerns of drug resistance. Preservation of existing drugs is necessary, as well as the development of new treatment options and methods of control. We focus this review on how the democratization of CRISPR/Cas9 genome editing technology can be enlisted to improve our understanding of the biology of nematode parasites and our ability to treat the infections they cause. We will first explore how this robust method of genome manipulation can be used to newly exploit the powerful model nematode Caenorhabditis elegans for parasitology research. We will then discuss potential avenues to develop CRISPR/Cas9 editing protocols in filarial nematodes. Lastly, we will propose potential ways in which CRISPR/Cas9 can be used to engineer gene drives that target the transmission of mosquito-borne filarial nematodes.
Lethal Gene Drive Selects Inbreeding
13627J. J. Bull, bioRxiv, 046847. 2016-06-03 13:55:41.
ere, population genetic models are used to consider the evolution of inbreeding (specifically selfing) as a possible response to a recessively lethal HEG with complete segregation distortion. Numerical analyses indicate a rich set of outcomes, but selfing often evolves in response to the HEG, with a corresponding partial restoration of mean fitness. Whether selfing does indeed evolve and its effect in restoring fitness depends heavily on the magnitude of inbreeding depression. Overall, these results point toward an underappreciated evolutionary response to block the harmful effects of a selfish gene.
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