Keywords: Replicator/site directed nuclease
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.
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 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.
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.
Guerrilla eugenics: gene drives in heritable human genome editing
26475A. D. Cutter, J Med Ethics, 2023-07-04 08:15:56.
CRISPR-Cas9 genome editing can and has altered human genomes, bringing bioethical debates about this capability to the forefront of philosophical and policy considerations. Here, I consider the underexplored implications of CRISPR-Cas9 gene drives for heritable human genome editing. Modification gene drives applied to heritable human genome editing would introduce a novel form of involuntary eugenic practice that I term guerrilla eugenics. Once introduced into a genome, stealth genetic editing by a gene drive genetic element would occur each subsequent generation irrespective of whether reproductive partners consent to it and irrespective of whether the genetic change confers any benefit. By overriding the ability to 'opt in' to genome editing, gene drives compromise the autonomy of carrier individuals and their reproductive partners to choose to use or avoid genome editing and impose additional burdens on those who hope to 'opt out' of further genome editing. High incidence of an initially rare gene drive in small human communities could occur within 200 years, with evolutionary fixation globally in a timeframe that is thousands of times sooner than achievable by non-drive germline editing. Following any introduction of heritable gene drives into human genomes, practices intended for surveillance or reversal also create fundamental ethical problems. Current policy guidelines do not comment explicitly on gene drives in humans. These considerations motivate an explicit moratorium as being warranted on gene drive development in heritable human genome editing.
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.
CRISPR-based gene editing of non-homologous end joining factors biases DNA repair pathway choice toward single-strand annealing in Aedes aegypti
25461K. Chae, J. M. Overcash, C. Dawson, C. Valentin, H. Tsujimoto, K. M. Myles and Z. N. Adelman, Current Research in Biotechnology, 5:100133. 2023-05-29 08:04:22.
To maintain genome stability, eukaryotic cells orchestrate DNA repair pathways to process DNA double-strand breaks (DSBs) that result from diverse developmental or environmental stimuli. Bias in the selection of DSB repair pathways, either non-homologous end joining (NHEJ) or homology-directed repair (HDR), is also critical for efficient gene editing and for homing-based gene drive approaches developed for the control of disease-transmitting vector mosquitoes. However, little is understood about DNA repair homeostasis in the mosquito genome. Here, we utilized CRISPR/Cas9 to generate indel mutant strains for core NHEJ factors ku80, DNA ligase IV (lig4), and DNA-PKcs in the mosquito Aedes aegypti and evaluated the corresponding effects on DNA repair. In a plasmid-based assay, disruption of ku80 or lig4, but not DNA-PKcs, reduced both NHEJ and SSA. However, a transgenic reporter strain-based test revealed that those mutations significantly biased DNA repair events toward SSA. Interestingly, ku80 mutation also significantly increased the end joining rate by a yet-characterized mechanism in males. Our study provides evidence that the core NHEJ factors have an antagonistic effect on SSA-based DSB repair of the Ae. aegypti genome. Down-modulating the NHEJ pathway can enhance the efficiency of nuclease-based genetic control approaches, as most of those operate by homology-based repair processes along with extensive DNA end resection that is antagonized by NHEJ.
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.
Evolution driven by genetic engineering should be known as ‘genetic welding’ to draw scientific and ethical scrutiny
24940S. Moore, AZO Life Sciences, 2023-03-29 10:13:27.
The advent of CRISPR-Cas9 technology has been revolutionary, but it has also been highly controversial. In an opinion paper published in the journal Trends in Genetics, evolutionary geneticist Asher Cutter highlights the importance of coining the term ‘genetic welding’ for the anthropogenic manipulation of genetic drive made possible by tools such as CRISPR-Cas9. Separating this process from other processes that influence evolution might be fundamental to ensuring proper consideration is given to the potential future outcomes of genetic welding.
Next-generation CRISPR gene-drive systems using Cas12a nuclease
24783S. Sanz Juste, E. M. Okamoto, X. Feng and V. L. Del Amo, bioRxiv, 2023.02.20.529271. 2023-02-20 09:40:53.
One method for reducing the impact of vector-borne diseases is through the use of CRISPR-based gene drives, which manipulate insect populations due to their ability to rapidly propagate desired genetic traits into a target population. However, all current gene drives employ a Cas9 nuclease that is constitutively active, impeding our control over their propagation abilities and limiting the generation of novel gene drive arrangements. Yet, other nucleases such as the temperature-sensitive Cas12a have not been explored for gene drive designs. To address this, we herein present a proof-of-concept gene-drive system driven by Cas12a that can be regulated via temperature modulation. Furthermore, we combined Cas9 and Cas12a to build double gene drives capable of simultaneously spreading two independent engineered alleles. The development of Cas12a-mediated gene drives provides an innovative option for designing next-generation vector control strategies to combat disease vectors and agricultural pests.Competing Interest StatementAll authors declare no competing interests.
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.
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.
Modeling the efficacy of CRISPR gene drive for snail immunity on schistosomiasis control
23810R. E. Grewelle, J. Perez-Saez, J. Tycko, E. K. O. Namigai, C. G. Rickards and G. A. De Leo, PLOS Neglected Tropical Diseases, 16:e0010894. 2022-10-31 09:02:03.
CRISPR gene drives could revolutionize the control of infectious diseases by accelerating the spread of engineered traits that limit parasite transmission in wild populations. Gene drive technology in mollusks has received little attention despite the role of freshwater snails as hosts of parasitic flukes causing 200 million annual cases of schistosomiasis. A successful drive in snails must overcome self-fertilization, a common feature of host snails which could prevents a drive's spread. Here we developed a novel population genetic model accounting for snails' mixed mating and population dynamics, susceptibility to parasite infection regulated by multiple alleles, fitness differences between genotypes, and a range of drive characteristics. We integrated this model with an epidemiological model of schistosomiasis transmission to show that a snail population modification drive targeting immunity to infection can be hindered by a variety of biological and ecological factors; yet under a range of conditions, disease reduction achieved by chemotherapy treatment of the human population can be maintained with a drive. Alone a drive modifying snail immunity could achieve significant disease reduction in humans several years after release. 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 evaluation of the genetic and ecological landscape.
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.
Anopheles homing suppression drive candidates exhibit unexpected performance differences in simulations with spatial structure
23734S. E. Champer, I. K. Kim, A. G. Clark, P. W. Messer and J. Champer, eLife, 11:e79121. 2022-10-14 06:37:55.
Recent experiments have produced several Anopheles gambiae homing gene drives that disrupt female fertility genes, thereby eventually inducing population collapse. Such drives may be highly effective tools to combat malaria. One such homing drive, based on the zpg promoter driving CRISPR/Cas9, was able to eliminate a cage population of mosquitoes. A second version, purportedly improved upon the first by incorporating an X-shredder element (which biases inheritance towards male offspring), was similarly successful. Here, we analyze experimental data from each of these gene drives to extract their characteristics and performance parameters and compare these to previous interpretations of their experimental performance. We assess each suppression drive within an individual-based simulation framework that models mosquito population dynamics in continuous space. We find that the combined homing/X-shredder drive is actually less effective at population suppression within the context of our mosquito population model. In particular, the combined drive often fails to completely suppress the population, instead resulting in an unstable equilibrium between drive and wild-type alleles. By contrast, otherwise similar drives based on the nos promoter may prove to be more promising candidates for future development than originally thought.
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.
A population modification gene drive targeting both Saglin and Lipophorin disables Plasmodium transmission in Anopheles mosquitoes
23150E. I. Green, E. Jaouen, D. Klug, R. P. Olmo, A. Gautier, S. A. Blandin and E. Marois, bioRxiv, 2022.07.08.499187. 2022-07-08 09:32:53.
Lipophorin is an essential, highly expressed lipid transporter protein that is secreted and circulates in insect hemolymph. We hijacked the Anopheles gambiae 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 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 hitch-hike together with the Saglin drive. Although producing drive-resistant alleles, 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 affect parasite transmission by 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.Competing Interest StatementThe authors have declared no competing interest.
Genetic Approaches for Controlling CRISPR-based Autonomous Homing Gene Drives
23115P. R. Chennuri, Z. N. Adelman and K. M. Myles, Frontiers in Bioengineering and Biotechnology, 10:897231. 2022-06-15 08:41:17.
CRISPR-based autonomous homing gene drives are a potentially transformative technology with the power to reduce the prevalence of, or even eliminate, vector-borne diseases, agricultural pests, and invasive species. However, there are a number of regulatory, ethical, environmental, and sociopolitical concerns surrounding the potential use of gene drives, particularly regarding the possibility for any unintended outcomes that might result from such a powerful technology. Therefore, there is an imminent need for countermeasures or technologies capable of exerting precise spatiotemporal control of gene drives, if their transformative potential is ever to be fully realized. This review summarizes the current state of the art in the development of technologies to prevent the uncontrolled spread of CRISPR-based autonomous homing 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.
Adversarial interspecies relationships facilitate population suppression by gene drive in spatially explicit models
22288Y. Liu, W. Teo, H. Yang and J. Champer, bioRxiv, 2022.05.08.491087. 2022-05-08 08:15:29.
Suppression gene drives are designed to bias their inheritance and increase in frequency in a population, disrupting an essential gene in the process. When the frequency is high enough, the population will be unable to reproduce above the replacement level and could be eliminated. CRISPR suppression drives based on the homing mechanism have already seen success in the laboratory, particularly in malaria mosquitoes. However, several models predict that the use of these drives in realistic populations with spatial structure may not achieve complete success. This is due to the ability of wild-type individuals to escape the drive and reach empty areas with reduced competition, allowing them to achieve high reproductive success and leading to extinction-recolonization cycles across the landscape. Here, we extend our continuous space gene drive framework to include two competing species or predator-prey species pairs. We find in both discrete-generation and mosquito-specific models that the presence of a competing species or predator can greatly facilitate drive-based suppression, even for drives with modest efficiency. However, the presence of a competing species also substantially increases the frequency of outcomes in which the drive is lost before suppression is achieved. These results are robust in models with seasonal population fluctuations. We also found that suppression can be somewhat more difficult if targeting a predator with strong predator-prey interactions. Our results illustrate the difficulty of predicting outcomes of interventions that could substantially affect the populations of interacting species in complex ecosystems. However, our results are also potentially promising for the prospects of less powerful gene drives in achieving successful elimination of target pest populations.Competing Interest StatementThe authors have declared no competing interest.
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.
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.
Finding the strongest gene drive: Simulations reveal unexpected performance differences between Anopheles homing suppression drive candidates
21566S. E. Champer, I. K. Kim, A. G. Clark, P. W. Messer and J. Champer, bioRxiv, 2022.03.28.486009. 2022-03-28 12:29:28.
Recent experiments have produced several Anopheles gambiae homing gene drives that disrupt female fertility genes, thereby eventually inducing population collapse. Such drives may be highly effective tools to combat malaria. One such homing drive, based on the zpg promoter driving CRISPR/Cas9, was able to eliminate a cage population of mosquitoes. A second version, purportedly improved upon the first by incorporating an X-shredder element (which biases inheritance towards male offspring), was similarly successful. Here, we re-analyze the data of each of these gene drives and suggest an alternative interpretation of their performance. We assess each suppression drive within an individual-based simulation framework that models mosquito population dynamics in continuous space. We find that the combined homing/X-shredder drive is actually less effective at population suppression within the context of our mosquito population model. In particular, the combined drive often fails to completely suppress the population, instead resulting in an unstable equilibrium between drive and wild-type alleles. By contrast, otherwise similar drives based on the nos promoter may prove to be more promising candidates for future development due to potentially superior performance.Competing Interest StatementThe authors have declared no competing interest.
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.
Gene drives and population persistence vs elimination: The impact of spatial structure and inbreeding at low density
20529P. J. Beaghton and A. Burt, Theoretical Population Biology, 2022-03-03 08:28:52.
Synthetic gene drive constructs are being developed to control disease vectors, invasive species, and other pest species. In a well-mixed random mating population a sufficiently strong gene drive is expected to eliminate a target population, but it is not clear whether the same is true when spatial processes play a role. In species with an appropriate biology it is possible that drive-induced reductions in density might lead to increased inbreeding, reducing the efficacy of drive, eventually leading to suppression rather than elimination, regardless of how strong the drive is. To investigate this question we analyse a series of explicitly solvable stochastic models considering a range of scenarios for the relative timing of mating, reproduction, and dispersal and analyse the impact of two different types of gene drive, a Driving Y chromosome and a homing construct targeting an essential gene. We find in all cases a sufficiently strong Driving Y will go to fixation and the population will be eliminated, except in the one life history scenario (reproduction and mating in patches followed by dispersal) where low density leads to increased inbreeding, in which case the population persists indefinitely, tending to either a stable equilibrium or a limit cycle. These dynamics arise because Driving Y males have reduced mating success, particularly at low densities, due to having fewer sisters to mate with. Increased inbreeding at low densities can also prevent a homing construct from eliminating a population. For both types of drive, if there is strong inbreeding depression, then the population cannot be rescued by inbreeding and it is eliminated. These results highlight the potentially critical role that low-density-induced inbreeding and inbreeding depression (and, by extension, other sources of Allee effects) can have on the eventual impact of a gene drive on a target population.
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-drive mosquitoes, a prospect for future malaria control
21770S. A. Monawwer, A. O. I. Alzubaidi, F. Yasmin, S. M. Q. Haimour, S. M. I. Shay and I. Ullah, Pan African Medical Journal, 41:2-6. 2022-02-08 07:58:25.
Despite major developments in malaria control over the past two decades, the disease continues to scourge the human population across the globe. Rising concerns such as insecticide resistance amongst vector mosquitoes are a cause of huge fear amongst healthcare providers and policymakers. Amidst such dire circumstances, a recent development may form the blueprint for future malaria control as for the first time ever researchers were able to decimate an entire mosquito population using gene-drive technology within a span of one year in a multi-generation, ecologically challenging study. Despite some concerns, the technology displayed a high potential of becoming a powerful tool in malaria control.
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.
Modeling CRISPR gene drives for suppression of invasive rodents using a supervised machine learning framework
19764S. E. Champer, N. Oakes, R. Sharma, P. García-Díaz, J. Champer and P. W. Messer, PLoS Comput Biol, 17:e1009660. 2021-12-29 13:01:20.
Invasive rodent populations pose a threat to biodiversity across the globe. When confronted with these invaders, native species that evolved independently are often defenseless. CRISPR gene drive systems could provide a solution to this problem by spreading transgenes among invaders that induce population collapse, and could be deployed even where traditional control methods are impractical or prohibitively expensive. Here, we develop a high-fidelity model of an island population of invasive rodents that includes three types of suppression gene drive systems. The individual-based model is spatially explicit, allows for overlapping generations and a fluctuating population size, and includes variables for drive fitness, efficiency, resistance allele formation rate, as well as a variety of ecological parameters. The computational burden of evaluating a model with such a high number of parameters presents a substantial barrier to a comprehensive understanding of its outcome space. We therefore accompany our population model with a meta-model that utilizes supervised machine learning to approximate the outcome space of the underlying model with a high degree of accuracy. This enables us to conduct an exhaustive inquiry of the population model, including variance-based sensitivity analyses using tens of millions of evaluations. Our results suggest that sufficiently capable gene drive systems have the potential to eliminate island populations of rodents under a wide range of demographic assumptions, though only if resistance can be kept to a minimal level. This study highlights the power of supervised machine learning to identify the key parameters and processes that determine the population dynamics of a complex evolutionary system.
Modeling impact and cost-effectiveness of driving-Y gene drives for malaria elimination in the Democratic Republic of the Congo
19594N. Metchanun, C. Borgemeister, G. Amzati, J. von Braun, M. Nikolov, P. Selvaraj and J. Gerardin, Evolutionary Applications, 2021-12-07 17:59:09.
Malaria elimination will be challenging in countries that currently continue to bear high malaria burden. Sex-ratio distorting gene drives, such as driving-Y, could play a role in an integrated elimination strategy if they can effectively suppress vector populations. Using a spatially explicit, agent-based model of malaria transmission in eight provinces spanning the range of transmission intensities across the Democratic Republic of the Congo, we predict the impact and cost-effectiveness of integrating driving-Y gene drive mosquitoes in malaria elimination strategies that include existing interventions such as insecticide-treated nets and case management of symptomatic malaria. Gene drive mosquitoes could eliminate malaria and were the most cost-effective intervention overall if the drive component was highly effective with at least 95% X-shredder efficiency at relatively low fertility cost, and associated cost of deployment below 7.17 $int per person per year. Suppression gene drive could be a cost-effective supplemental intervention for malaria elimination, but tight constraints on drive effectiveness and cost ceilings may limit its feasibility.
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 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.
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.
Autocatalytic-protection for an unknown locus CRISPR-Cas countermeasure for undesired mutagenic chain reactions
17969E. Schonfeld, E. Schonfeld and D. Schonfeld, Journal of Theoretical Biology, 528:110831. 2021-07-11 13:41:33.
The mutagenic chain reaction (MCR) is a genetic tool to use a CRISPR–Cas construct to introduce a homing endonuclease, allowing gene drive to influence whole populations in a minimal number of generations (Esvelt et al., 2014, Gantz and Bier, 2015, Gantz and Bier, 2016). The question arises: if an active genetic terror event is released into a population, could we prevent the total spread of the undesired allele (Gantz, et al., 2015, Webber et al., 2015)? Thus far, effective protection methods require knowledge of the terror locus (Grunwald et al., 2019). Here we introduce a novel approach, an autocatalytic-Protection for an Unknown Locus (a-PUL), whose aim is to spread through a population and arrest and decrease an active terror event’s spread without any prior knowledge of the terror-modified locus, thus allowing later natural selection and ERACR drives to restore the normal locus (Hammond et al., 2017). a-PUL, using a mutagenic chain reaction, includes (i) a segment encoding a non-Cas9 endonuclease capable of homology-directed repair suggested as Type II endonuclease Cpf1 (Cas12a), (ii) a ubiquitously-expressed gene encoding a gRNA (gRNA1) with a U4AU4 3′-overhang specific to Cpf1 and with crRNA specific to some desired genomic sequence of non-coding DNA, (iii) a ubiquitously-expressed gene encoding two gRNAs (gRNA2/gRNA3) both with tracrRNA specific to Cas9 and crRNA specific to two distinct sites of the Cas9 locus, and (iv) homology arms flanking the Cpf1/gRNA1/gRNA2/gRNA3 cassette that are identical to the region surrounding the target cut directed by gRNA1 (Khan, 2016, Zetsche et al., 2015). We demonstrate the proof-of-concept and efficacy of our protection construct through a Graphical Markov model and computer simulation.
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).
Experimental demonstration of tethered gene drive systems for confined population modification or suppression
17153M. Metzloff, E. Yang, S. Dhole, A. G. Clark, P. W. Messer and J. Champer, bioRxiv, 2021.05.29.446308. 2021-05-30 10:50:23.
Tethered drive systems, in which a locally confined gene drive provides the CRISPR nuclease needed for a homing drive, could provide a solution to this problem, offering the power of a homing drive and confinement of the supporting drive. Here, we demonstrate the engineering of a tethered drive system in Drosophila, using a TARE drive to support modification and suppression homing drives. Each drive was able to bias inheritance in its favor, and the TARE drive was shown to spread only when released above a threshold frequency in experimental cage populations. After the TARE drive had established in the population, it facilitated the spread of a subsequently released split homing modification drive (to all individuals in the cage) and of a homing suppression drive (to its equilibrium frequency). Our results show that the tethered drive strategy is a viable and easily engineered option for providing confinement of homing drives to target populations. Competing Interest StatementThe authors have declared no competing interest.
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.
Small-Cage Laboratory Trials of Genetically-Engineered Anopheline Mosquitoes
17068R. Carballar-Lejarazú, T. B. Pham, V. Bottino-Rojas, A. Adolfi and A. A. James, J Vis Exp, 2021-05-18 14:30:01.
Control of mosquito-borne pathogens using genetically-modified vectors has been proposed as a promising tool to complement conventional control strategies. CRISPR-based homing gene drive systems have made transgenic technologies more accessible within the scientific community. Evaluation of transgenic mosquito performance and comparisons with wild-type counterparts in small laboratory cage trials provide valuable data for the design of subsequent field cage experiments and experimental assessments to refine the strategies for disease prevention. Here, we present three different protocols used in laboratory settings to evaluate transgene spread in anopheline mosquito vectors of malaria. These include inundative releases (no gene-drive system), and gene-drive overlapping and non-overlapping generation trials. The three trials vary in a number of parameters and can be adapted to desired experimental settings. Moreover, insectary studies in small cages are part of the progressive transition of engineered insects from the laboratory to open field releases. Therefore, the protocols described here represent invaluable tools to provide empirical values that will ultimately aid field implementation of new technologies for malaria elimination.
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.
Gene-Editing Approach To Control the Invasive Gray Squirrel
16572M. Campbell, Technology Networks, 2021-03-08 14:33:09.
Biodiversity refers to the extent of the variety of life that is found on planet Earth – and it is currently under threat. Changes in biodiversity have been flagged as "surpassing safe limits" for several years, and world leaders and scientists across the globe are consequently exploring different ways to address the crisis. Invasive species, defined by National Geographic as "an organism that is not indigenous, or native, to a particular area", threaten planet Earth's biodiversity to an even greater extent than climate change. The Intergovernmental Science-Policy Platform on Biodiversity and Ecosystem Services (IPBES) Global Assessment Report found that the number of invasive species per country have risen by ~70% since 1970 across 21 countries that were included in the report. An example of an invasive species is the gray squirrel Sciurus carolinensis found in the UK, which outcompetes the native red squirrel for resources and habitats and carries diseases that are not pathogenic to themselves but can kill red squirrels.
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.
Ecology: Gene drives may help control invasive grey squirrel in the UK
16557A. Korn, EurekaAlert, 2021-03-04 19:50:50.
Gene drives introduce genes into a population that have been changed to induce infertility in females, allowing for the control of population size. However, they face technical challenges, such as controlling the spread of altered genes as gene drive individuals mate with wild individuals, and the development of genetic resistance, which may render the gene drive ineffective. To address these challenges, Nicky Faber and colleagues used computer modelling to investigate the effectiveness of a combination of three gene drive technologies using the grey squirrel as a case study.
Genetically modified squirrels could curb growing population of greys
16542S. Knapton, Telegraph, 2021-03-04 16:06:59.
Mutant grey squirrels, genetically modified to spread infertility genes, could be released into the wild to tackle the burgeoning population,
Expert reaction to a paper suggesting that gene drives could be used to help control grey squirrel numbers in the UK
16540Anonymous, Science Media Centre, 2021-03-04 15:59:10.
This study assesses the prospects for using a gene drive to control invasive grey squirrels in the UK. This is a modelling study exploring the potential for such an approach – no such gene drives currently exist and developing them for grey squirrels would be quite a long-term project. Invasive species are a major problem for biodiversity and conservation; in many cases there are no adequate methods for control. Genetic methods may provide new options, potentially both more effective and with fewer off-target effects. Most work on gene drives has focused on mosquitoes; this study is unusual in focusing on a vertebrate (grey squirrel), though there has also been interest in targeting invasive populations of mice and rats on islands.
CRISPR gene drives may come to a squirrel near you.
16536Anonymous, NewsBeezer, 2021-03-04 15:48:43.
Today’s gene drive technologies could be blended to provide control of the invasive gray squirrel population in the UK – with minimal risk to other populations, according to a new modeling published in the journal Scientific reports. Gene driving introduces altered genes into a population that can cause infertility in women. This allows scientists to control the size of the population. However, this tactic faces technical difficulties such as controlling the spread of altered genes while certain animals that are part of the gene drive population mate with uncontrolled populations ̵
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.
Modeling impact and cost-effectiveness of gene drives for malaria elimination in the Democratic Republic of the Congo
16477N. Metchanun, C. Borgemeister, G. Amzati, J. von Braun, M. Nikolov, P. Selvaraj and J. Gerardin, medRxiv, 2020.06.29.20142760. 2021-02-22 13:51:04.
Using a spatially explicit, agent-based model of malaria transmission in eight representative provinces of the Democratic Republic of the Congo, we predict the impact and cost-effectiveness of integrating driving-Y gene drive mosquitoes in malaria elimination strategies that include existing interventions such as insecticide-treated nets and case management of symptomatic malaria. Gene drive mosquitoes could eliminate malaria and were the most cost-effective intervention overall if the drive component was highly effective with at least 95% X-shredding and associated cost of deployment below 7.17 $int per person per year. Suppression gene drive could be a cost-effective supplemental intervention for malaria elimination, but tight constraints on drive effectiveness and cost ceilings may limit its feasibility.
Regulating the expression of gene drives is key to increasing their invasive potential and the mitigation of resistance
16290A. Hammond, X. Karlsson, I. Morianou, K. Kyrou, A. Beaghton, M. Gribble, N. Kranjc, R. Galizi, A. Burt, A. Crisanti and T. Nolan, PLOS Genetics, 17:e1009321. 2021-01-29 21:53:25.
Here we show that restricting the cutting activity of the gene drive to the germline tissue is crucial to maintaining its potency and we illustrate how failure to restrict this activity can lead to the generation of mutations that can make mosquitoes resistant to the gene drive.
Demographic feedbacks can hamper the spatial spread of a gene drive
16203L. Girardin and F. Débarre, arXiv, 2021-01-27 15:32:53.
Our results indicate that taking into account the interplay between population dynamics and population genetics might actually be crucial, as it can effectively reverse the direction of the invasion and lead to failure. Our findings can be extended to other bistable systems, such as the spread of cytoplasmic incompatibilities caused by Wolbachia.
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'.
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.
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.
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: 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%.
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.
MGDrivE 2: A simulation framework for gene drive systems incorporating seasonality and epidemiological dynamics
14733S. L. Wu, J. B. Bennett, H. M. Sanchez C, A. J. Dolgert, T. M. Leon and J. M. Marshall, bioRxiv, 2020.10.16.343376. 2020-10-17 15:42:27.
We present MGDrivE 2 (Mosquito Gene Drive Explorer 2): an extension of and development from the MGDrivE 1 simulation framework that investigates the population dynamics of a variety of gene drive architectures and their spread through spatially-explicit mosquito populations.
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.
Engineering the Composition and Fate of Wild Populations with Gene Drive
14731B. A. Hay, G. Oberhofer and M. Guo, Annual Review of Entomology, 2020-10-09 15:37:03.
We describe technologies under consideration, progress that has been made, and remaining technological hurdles, particularly with respect to evolutionary stability and our ability to control the spread and ultimate fate of genes introduced into populations.
Assessing the acoustic behaviour of Anopheles gambiae (s.l.) dsxF mutants: implications for vector control
14647M. P. Su, M. Georgiades, J. Bagi, K. Kyrou, A. Crisanti and J. T. Albert, Parasites and Vectors, 13:507. 2020-10-07 13:33:23.
We analysed sound emissions and acoustic preference in a doublesex mutant previously used to collapse Anopheles gambiae (s.l.) cages.
Gene Drive Control Worry Eased by Genetic Neutralizing Elements
14452Staff, Genetic Engineering and Biotechnology News, 2020-09-21 17:17:09.
Bier and his colleagues have developed two new active genetic systems that address such risks by halting or eliminating gene drives in the wild, offering two new solutions based on elements developed in the common fruit fly. T
Active Genetic Neutralizing Elements for Halting or Deleting Gene Drives
14444X.-R. S. Xu, E. A. Bulger, V. M. Gantz, C. Klanseck, S. R. Heimler, A. Auradkar, J. B. Bennett, L. A. Miller, S. Leahy, S. S. Juste, A. Buchman, O. S. Akbari, J. M. Marshall and E. Bier, Molecular Cell, 2020-09-18 11:30:45.
Here we describe two self-copying (or active) guide RNA-only genetic elements, called e-CHACRs and ERACRs. These elements use Cas9 produced in trans by a gene drive either to inactivate the cas9 transgene (e-CHACRs) or to delete and replace the gene drive (ERACRs).
Biologists create new genetic systems to neutralize gene drives
14442University of California San Diego, ScienceDaily, 2020-09-18 11:24:50.
Now, scientists at the University of California San Diego and their colleagues have developed two new active genetic systems that address such risks by halting or eliminating gene drives in the wild.
A CRISPR homing gene drive targeting a haplolethal gene removes resistance alleles and successfully spreads through a cage population
14387J. Champer, E. Yang, E. Lee, J. Liu, A. G. Clark and P. W. Messer, Proceedings of the National Academy of Sciences, 202004373. 2020-09-14 15:23:07.
Here, we present a CRISPR homing drive that was able to successfully spread to all individuals in a laboratory cage study in Drosophila melanogaster without any apparent evolution of resistance.
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
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.
Novel combination of CRISPR-based gene drives eliminates resistance and localises spread
14172N. R. Faber, G. R. McFarlane, R. C. Gaynor, I. Pocrnic, C. B. A. Whitelaw and G. Gorjanc, bioRxiv, 2020-08-27 14:22:46.
We present HD-ClvR, a novel combination of CRISPR-based gene drives that eliminates resistance and localises spread. As a case study, we model HD-ClvR in the grey squirrel (Sciurus carolinensis), which is an invasive pest in the UK and responsible for both biodiversity and economic losses.
Anthony James / Mosquito Modification
14229Big Picture Science, SETI Institute, 2020-08-24 20:26:09.
Anthony James, vector biologist at the University of California, Irvine, describeshow we might genetically modify mosquitoes to make them unable to pass malaria on to humans.
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.
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.
CRISPR gene drives could eliminate many vector-driven pests and diseases, but challenges remain
13631J. Champer, Genetic Literacy Project, 2020-08-06 14:11:01.
A functioning gene drive system could fundamentally change our strategies for the control of vector-borne diseases by facilitating rapid dissemination of transgenes that prevent pathogen transmission or reduce vector capacity. CRISPR/Cas9 gene drive promises such a mechanism, which works by converting cells that are heterozygous for the drive construct into homozygotes, thereby enabling super-Mendelian inheritance.
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.
Chemical controllable gene drive in Drosophila
13773D. Chae, J. Lee, N. Lee, K. Park, S. J. Moon and H. H. Kim, ACS Synthetic Biology, in press. 2020-07-30 12:48:10.
Here, we report a chemical-induced control of gene drive. We prepared a CRISPR-based gene drive system that can be removed by a site-specific recombinase, Rippase, the expression of which is induced by the chemical RU486 in fruit flies. Exposure of fruit flies to RU486 resulted in 7-12% removal of gene drive elements at each generation, leading to a significant reduction in gene drive-fly propagation.
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.
Detecting the population dynamics of an autosomal sex ratio distorter transgene in malaria vector mosquitoes
13607P. Pollegioni, A. R. North, T. Persampieri, A. Bucci, R. L. Minuz, D. A. Groneberg, T. Nolan, P. A. Papathanos, A. Crisanti and R. Muller, Journal of Applied Ecology, 11. 2020-06-18 12:44:16.
A sex-distorting autosomal transgene has been developed recently in G3 mosquitoes, a laboratory strain of the malaria vectorAnopheles gambiaes.l. Following the World Health Organization guidance framework for the testing of GM mosquitoes, we assessed the dynamics of this transgene in large cages using a joint experimental modelling approach. We performed a 4-month experiment in large, indoor cages to study the population genetics of the transgene.
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.
The yeast mating-type switching endonuclease HO is a domesticated member of an unorthodox homing genetic element family
11244A. Y. Coughlan, L. Lombardi, S. Braun-Galleani, A. A. R. Martos, V. Galeote, F. Bigey, S. Dequin, K. P. Byrne and K. H. Wolfe, eLife, 9:e55336. 2020-04-27 17:30:45.
The mating-type switching endonuclease HO plays a central role in the natural life cycle of Saccharomyces cerevisiae, but its evolutionary origin is unknown. HO is a recent addition to yeast genomes, present in only a few genera close to Saccharomyces. Here we show that HO is structurally and phylogenetically related to a family of unorthodox homing genetic elements found in Torulaspora and Lachancea yeasts. These WHO elements home into the aldolase gene FBA1, replacing its 3' end each time they integrate. They resemble inteins but they operate by a different mechanism that does not require protein splicing. We show that a WHO protein cleaves Torulaspora delbrueckii FBA1 efficiently and in an allele-specific manner, leading to DNA repair by gene conversion or NHEJ. The DNA rearrangement steps during WHO element homing are very similar to those during mating-type switching, and indicate that HO is a domesticated WHO-like element.
Strategies for Achieving Gene Drive – Over-Replication
11404GeneConvene Global Collaborative, 2020-03-24 13:10:14.
This short video explains how there are essentially three genetic strategies for achieve 'drive' or 'gene drive'. This video focuses on Over-Replication, a very common strategy associated wtih different types of genetic elements found in nature that 'drive'. This strategy is also used by researchers and developers to assemble transgenes that drive when introduced into the genome of an organism.
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.
Performance analysis of novel toxin-antidote CRISPR gene drive systems
7969J. Champer, I. K. Kim, S. E. Champer, A. G. Clark and P. W. Messer, BMC Biology, 18:27. 2020-03-12 14:48:01.
CRISPR gene drive systems allow the rapid spread of a genetic construct throughout a population. Such systems promise novel strategies for the management of vector-borne diseases and invasive species by suppressing a target population or modifying it with a desired trait. However, current homing-type drives have two potential shortcomings. First, they can be thwarted by the rapid evolution of resistance. Second, they lack any mechanism for confinement to a specific target population. In this study, we conduct a comprehensive performance assessment of several new types of CRISPR-based gene drive systems employing toxin-antidote (TA) principles, which should be less prone to resistance and allow for the confinement of drives to a target population due to invasion frequency thresholds.
Gene technologies in weed management: a technical feasibility analysis
7948N. Kumaran, A. Choudhary, M. Legros, A. W. Sheppard, L. G. Barrett, D. M. Gardiner and S. Raghu, Current Opinion in Insect Science, 38:6-14. 2020-01-30 20:44:15.
With the advent of new genetic technologies such as gene silencing and gene drive, efforts to develop additional management tools for weed management is gaining significant momentum. These technologies promise novel ways to develop sustainable weed control options because gene silencing can switch-off genes mediating adaptation (e.g. growth, herbicide resistance), and gene drive can be used to spread modified traits and to engineer wild populations with reduced fitness. However, applying gene silencing and/or gene drive is expected to be inherently complex as their application is constrained by several methodological and technological difficulties. In this review we explore the challenges of these technologies, and discuss strategies and resources accessible to accelerate the development of gene-tech based tools for weed management. We also highlight how gene technologies can be integrated into existing management tactics such as classical biological control, and their possible interactions.
A transcomplementing gene drive provides a flexible platform for laboratory investigation and potential field deployment
6668V. López Del Amo, A. L. Bishop, H. M. Sánchez C, J. B. Bennett, X. Feng, J. M. Marshall, E. Bier and V. M. Gantz, Nature Communications, 11:352. 2020-01-17 18:00:23.
CRISPR-based gene drives can spread through wild populations by biasing their own transmission above the 50% value predicted by Mendelian inheritance. These technologies offer population-engineering solutions for combating vector-borne diseases, managing crop pests, and supporting ecosystem conservation efforts. Current technologies raise safety concerns for unintended gene propagation. Herein, we address such concerns by splitting the drive components, Cas9 and gRNAs, into separate alleles to form a trans-complementing split–gene-drive (tGD) and demonstrate its ability to promote super-Mendelian inheritance of the separate transgenes. This dual-component configuration allows for combinatorial transgene optimization and increases safety by restricting escape concerns to experimentation windows. We employ the tGD and a small–molecule-controlled version to investigate the biology of component inheritance and resistant allele formation, and to study the effects of maternal inheritance and impaired homology on efficiency. Lastly, mathematical modeling of tGD spread within populations reveals potential advantages for improving current gene-drive technologies for field population modification.
The potential for a released autosomal X-shredder becoming a driving-Y chromosome and invasively suppressing wild populations of malaria mosquitoes
5648Alcalay, Y., S. Fuchs, R. Galizi, F. Bernardini, R. E. Haghighat-Khah, D. B. Rusch, J. R. Adrion, M. W. Hahn, P. Tortosa and P. A. Papathanos, bioRxiv, 2019:860551. 2019-12-17 17:51:02.
Synthetic sex-ratio distorters based on X-chromosome shredding are predicted to be more efficient than sterile males for population suppression of malaria mosquitoes using genetic control. X chromosome shredding operates through the targeted elimination of X-chromosome-bearing gametes during male spermatogenesis, resulting in males that have a high fraction of male offspring. Strains harboring autosomal constructs containing a modified endonuclease I-PpoI have now been developed in the malaria mosquito Anopheles gambiae, resulting in strong sex-ratio distortion towards males. Data are being gathered for these strains for submission of regulatory dossiers for contained use and subsequent field release in West Africa. Since autosomal X shredders are transmitted in a Mendelian fashion and can be selected against their frequency in the population is expected to decline once releases are halted. However, any unintended transfer of the X-shredder to the Y-chromosome could theoretically change these dynamics: This could lead to 100% transmission of the newly Y-linked X-shredder to the predominant male-biased offspring and its insulation from negative selection in females, resulting in its potential spread in the population and ultimately to suppression. Here, we analyze plausible mechanisms whereby an autosomal X-shredder could become linked to the Y-chromosome after release and provide data regarding its potential for activity should it become linked to the Y-chromosome. Our results strongly suggest that Y-chromosome linkage through remobilization of the transposon used for the initial genetic transformation is unlikely, and that, in the unexpected event that the X-shredder becomes linked to the Y-chromosome, expression and activity of the X-shredder would likely be inhibited by meiotic sex chromosome inactivation. We conclude that a functioning X-shredding based Y-drive resulting from a naturally induced transposition or translocation of the transgene onto the Y-chromosome is unlikely.
Plasmodium falciparum (Haemosporodia: Plasmodiidae) and O’nyong-nyong virus development in a transgenic Anopheles gambiae (Diptera: Culicidae) strain
5634Mumford, J. D., C. A. Long, S. C. Weaver, K. Miura, E. Wang, R. Rotenberry, E. M. Dotson and M. Q. Benedict, " Journal of Medical Entomology, 56:936-941. 2019-12-17 16:55:10.
ransgenic Anopheles gambiae Giles (Diptera: Culicidae) mosquitoes have been developed that confer sexual sterility on males that carry a transgene encoding a protein which cuts ribosomal DNA. A relevant risk concern with transgenic mosquitoes is that their capacity to transmit known pathogens could be greater than the unmodified form. In this study, the ability to develop two human pathogens in these transgenic mosquitoes carrying a homing endonuclease which is expressed in the testes was compared with its nontransgenic siblings. Infections were performed with Plasmodium falciparum (Welch) and o’nyong-nyong virus (ONNV) and the results between the transgenic and nontransgenic sibling females were compared. There was no difference observed with ONNV isolate SG650 in intrathoracic infections or the 50% oral infectious dose measured at 14 d postinfection or in mean body titers. Some significant differences were observed for leg titers at the medium and highest doses for those individuals in which virus titer could be detected. No consistent difference was observed between the transgenic and nontransgenic comparator females in their ability to develop P. falciparum NF54 strain parasites. This particular transgene caused no significant effect in the ability of mosquitoes to become infected by these two pathogens in this genetic background. These results are discussed in the context of risk to human health if these transgenic individuals were present in the environment.
Viral gene drive in herpesviruses
5917Walter, M. and E. Verdin, bioRxiv, 2019:717017. 2019-07-30 16:38:14.
Herpesviruses are ubiquitous pathogens in need of novel therapeutic solutions. Current engineered gene drive strategies rely on sexual reproduction, and are thought to be restricted to sexual organisms. Here, we report on the design of a novel gene drive system that allows the spread of an engineered trait in populations of DNA viruses and, in particular, herpesviruses. We describe the successful transmission of a gene drive sequence between distinct strains of human cytomegalovirus (human herpesvirus 5) and show that gene drive viruses can efficiently target and replace wildtype populations in cell culture experiments. Our results indicate that viral gene drives can be used to suppress a viral infection and may represent a novel therapeutic strategy against herpesviruses.
Assessment of a split homing based gene drive for efficient knockout of multiple genes
5915Kandul, N. P., J. Liu, A. Buchman, V. M. Gantz, E. Bier and O. S. Akbari, bioRxiv, 2019:706929. 2019-07-18 16:34:25.
Homing based gene drives (HGD) possess the potential to spread linked cargo genes into natural populations and are poised to revolutionize population control of animals. Given that host-encoded genes have been identified that are important for pathogen transmission, targeting these genes using guide RNAs as cargo genes linked to drives may provide a robust method to prevent transmission. However, effectiveness of the inclusion of additional guide RNAs that target separate host encoded genes has not been thoroughly explored. To test this approach, here we generated a split-HGD in Drosophila melanogaster that encoded a drive linked effector consisting of a second gRNA engineered to target a separate host encoded gene, which we term a gRNA-mediated effector (GME). This design enabled us to assess homing and knockout efficiencies of two target genes simultaneously, and also explore the timing and tissue specificity of Cas9 expression on cleavage/homing rates. We demonstrate that inclusion of a GME can result in high efficiency of disruption of its target gene during super-Mendelian propagation of split-HGD. However, maternal deposition and embryonic expression of Cas9 resulted in the generation of drive resistant alleles which can accumulate and limit the spread of such a drive. Alternative design principles are discussed that could mitigate the accumulation of resistance alleles while incorporating a GME.
Daisy-chain gene drives
5497MIT Media Lab, 2019-04-03 18:36:17.
Who should decide whether, when, and how to alter the environment? These are hard questions, especially when the decisions will impact people in many different communities or nations. Daisy drive systems may help by empowering local communities to make decisions concerning their local environments without imposing them on anyone else.
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.
Dr. Kevin Esvelt, MIT | Gene Drive
5482iBiology, 2019-01-15 18:05:30.
Dr. Kevin Esvelt explains how CRISPR-based gene drives can be used to spread genetic alterations through wild populations. He discusses strategies to maximize benefit and minimize risk.
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.
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.
Gene drive to reduce malaria transmission in sub-Saharan Africa
3963Burt, AC, Mamadou; Crisanti, Andrea; Diabate, Abdoulaye; Kayondo, Jonathan K., Journal of Responsible Innovation, 5:S66-S80. 2018-01-21 00:00:00.
Despite impressive progress, malaria continues to impose a substantial burden of mortality and morbidity, particularly in sub-Saharan Africa, and new tools will be needed to achieve elimination. Gene drive is a natural process by which some genes are inherited at a greater-than-Mendelian rate and can spread through a population even if they cause harm to the organisms carrying them. Many different synthetic gene drive systems have been proposed to suppress the number of mosquitoes and/or reduce vector competence. As with any control measure, due attention should be paid to the possible evolution of resistance. No gene drive construct has yet been reported that is "field-ready" for release, and when such constructs are developed, they should be assessed on a case-by-case basis. Gene drive approaches to vector control promise to have a number of key features that motivate their continued development, and scrutiny, by all concerned.
Selfish genetic elements
3955Agren, JAC, A. G., PLOS Genetics, 14:20. 2018-01-13 00:00:00.
Selfish genetic elements (historically also referred to as selfish genes, ultra-selfish genes, selfish DNA, parasitic DNA, genomic outlaws) are genetic segments that can enhance their own transmission at the expense of other genes in the genome, even if this has no or a negative effect on organismal fitness. [1-6] Genomes have traditionally been viewed as cohesive units, with genes acting together to improve the fitness of the organism. However, when genes have some control over their own transmission, the rules can change, and so just like all social groups, genomes are vulnerable to selfish behaviour by their parts. Early observations of selfish genetic elements were made almost a century ago, but the topic did not get widespread attention until several decades later. Inspired by the gene-centred views of evolution popularized by George Williams[7] and Richard Dawkins,[8] two papers were published back-to-back in Nature in 1980-by Leslie Orgel and Francis Crick[9] and Ford Doolittle and Carmen Sapienza[10] respectively-introducing the concept of selfish genetic elements (at the time called "selfish DNA") to the wider scientific community. Both papers emphasized that genes can spread in a population regardless of their effect on organismal fitness as long as they have a transmission advantage. Selfish genetic elements have now been described in most groups of organisms, and they demonstrate a remarkable diversity in the ways by which they promote their own transmission.[11] Though long dismissed as genetic curiosities, with little relevance for evolution, they are now recognized to affect a wide swath of biological processes, ranging from genome size and architecture to speciation.[12]
A CRISPR–Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes
3987Kyrou, KH, Andrew M.; Galizi, Roberto; Kranjc, Nace; Burt, Austin; Beaghton, Andrea K.; Nolan, Tony; Crisanti, Andrea, Nature Biotechnology, 36:1062–1066. 2018-01-05 00:00:00.
In the human malaria vector Anopheles gambiae, the gene doublesex (Agdsx) encodes two alternatively spliced transcripts, dsx-female (AgdsxF) and dsx-male (AgdsxM), that control differentiation of the two sexes. The female transcript, unlike the male, contains an exon (exon 5) whose sequence is highly conserved in all Anopheles mosquitoes so far analyzed. We found that CRISPR–Cas9-targeted disruption of the intron 4–exon 5 boundary aimed at blocking the formation of functional AgdsxF did not affect male development or fertility, whereas females homozygous for the disrupted allele showed an intersex phenotype and complete sterility. A CRISPR–Cas9 gene drive construct targeting this same sequence spread rapidly in caged mosquitoes, reaching 100% prevalence within 7–11 generations while progressively reducing egg production to the point of total population collapse. Owing to functional constraint of the target sequence, no selection of alleles resistant to the gene drive occurred in these laboratory experiments. Cas9-resistant variants arose in each generation at the target site but did not block the spread of the drive.
Redkmer: An assembly-free pipeline for the identification of abundant and specific X-chromosome target sequences for X-shredding by CRISPR endonucleases
4006Papathanos, PAW, Nikolai, CRISPR Journal, 1:88-98. 2018-01-04 00:00:00.
CRISPR-based synthetic sex ratio distorters, which operate by shredding the X-chromosome during male meiosis, are promising tools for the area-wide control of harmful insect pest or disease vector species. X-shredders have been proposed as tools to suppress insect populations by biasing the sex ratio of the wild population toward males, thus reducing its natural reproductive potential. However, to build synthetic X-shredders based on CRISPR, the selection of gRNA targets, in the form of high-copy sequence repeats on the X chromosome of a given species, is difficult, since such repeats are not accurately resolved in genome assemblies and cannot be assigned to chromosomes with confidence. We have therefore developed the redkmer computational pipeline, designed to identify short and highly abundant sequence elements occurring uniquely on the X chromosome. Redkmer was designed to use as input minimally processed whole genome sequence data from males and females. We tested redkmer with short- and long-read whole genome sequence data of Anopheles gambiae, the major vector of human malaria, in which the X-shredding paradigm was originally developed. Redkmer established long reads as chromosomal proxies with excellent correlation to the genome assembly and used them to rank X-candidate kmers for their level of X-specificity and abundance. Among these, a high-confidence set of 25-mers was identified, many belonging to previously known X-chromosome repeats of Anopheles gambiae,including the ribosomal gene array and the selfish elements harbored within it. Data from a control strain, in which these repeats are shared with the Y chromosome, confirmed the elimination of these kmers during filtering. Finally, we show that redkmer output can be linked directly to gRNA selection and off-target prediction. In addition, the output of redkmer, including the prediction of chromosomal origin of single-molecule long reads and chromosome specific kmers, could also be used for the characterization of other biologically relevant sex chromosome sequences, a task that is frequently hampered by the repetitiveness of sex chromosome sequence content.
Behavior of homing endonuclease gene drives targeting genes required for viability or female fertility with multiplexed guide RNAs
4005Oberhofer, GI, Tobin; Hay, Bruce A., Proceedings of the National Academy of Sciences of the United States of America, 115:e9343. 2018-01-03 00:00:00.
Homing endonuclease gene (HEG)-based gene drive can bring about population suppression when genes required for viability or fertility are targeted. However, these strategies are vulnerable to failure through mechanisms that create alleles resistant to cleavage but that retain wild-type gene function. We show that resistance allele creation can be prevented through the use of guide RNAs designed to cleave a gene at four target sites. However, homing rates were modest, and the HEGs were unstable during homing. In addition, use of a promoter active in the female germline resulted in levels of HEG carryover that compromised the viability or fertility of HEG-bearing heterozygotes, thereby preventing drive. We propose strategies that can help to overcome these problems in next-generation HEG systems.A gene drive method of particular interest for population suppression utilizes homing endonuclease genes (HEGs), wherein a site-specific, nuclease-encoding cassette is copied, in the germline, into a target gene whose loss of function results in loss of viability or fertility in homozygous, but not heterozygous, progeny. Earlier work in Drosophila and mosquitoes utilized HEGs consisting of Cas9 and a single guide RNA (gRNA) that together target a specific gene for cleavage. Homing was observed, but resistant alleles immune to cleavage, while retaining wild-type gene function, were also created through nonhomologous end joining. Such alleles prevent drive and population suppression. Targeting a gene for cleavage at multiple positions has been suggested as a strategy to prevent the appearance of resistant alleles. To test this hypothesis, we generated two suppression HEGs in Drosophila melanogaster targeting genes required for embryonic viability or fertility, using a HEG consisting of CRISPR/Cas9 and gRNAs designed to cleave each gene at four positions. Rates of target locus cleavage were very high, and multiplexing of gRNAs prevented resistant allele formation. However, germline homing rates were modest, and the HEG cassette was unstable during homing events, resulting in frequent partial copying of HEGs that lacked gRNAs, a dominant marker gene, or Cas9. Finally, in drive experiments, the HEGs failed to spread due to the high fitness load induced in offspring as a result of maternal carryover of Cas9/gRNA complex activity. Alternative design principles are proposed that may mitigate these problems in future gene drive engineering.
Gene drive: Evolved and synthetic
3964Burt, AC, Andrea, ACS Chemical Biology, 13:343-346. 2018-01-02 00:00:00.
Drive is a process of accelerated inheritance from one generation to the next that allows some genes to spread rapidly through populations even if they do not contribute to—or indeed even if they detract from—organismal survival and reproduction. Genetic elements that can spread by drive include gametic and zygotic killers, meiotic drivers, homing endonuclease genes, B chromosomes, and transposable elements. The fact that gene drive can lead to the spread of fitness-reducing traits (including lethality and sterility) makes it an attractive process to consider exploiting to control disease vectors and other pests. There are a number of efforts to develop synthetic gene drive systems, particularly focused on the mosquito-borne diseases that continue to plague us.
Gene drive for Malaria control | Andrea Crisanti |
5519TEDx, 2017-04-04 19:23:37.
Andrea discusses his team's laboratory work that has developed a revolutionary technology to spread genetic modifications from few laboratory mosquitoes to wild populations to eradicate malaria in the near future
How driving endonuclease genes can be used to combat pests and disease vectors
4043Godfray, HCJN, A.; Burt, A., BMC Biology, 15:81. 2017-01-21 00:00:00.
Driving endonuclease genes (DEGs) spread through a population by a non-Mendelian mechanism. In a heterozygote, the protein encoded by a DEG causes a double-strand break in the homologous chromosome opposite to where its gene is inserted and when the break is repaired using the homologue as a template the DEG heterozygote is converted to a homozygote. Some DEGs occur naturally while several classes of endonucleases can be engineered to spread in this way, with CRISPR-Cas9 based systems being particularly flexible. There is great interest in using driving endonuclease genes to impose a genetic load on insects that vector diseases or are economic pests to reduce their population density, or to introduce a beneficial gene such as one that might interrupt disease transmission. This paper reviews both the population genetics and population dynamics of DEGs. It summarises the theory that guides the design of DEG constructs intended to perform different functions. It also reviews the studies that have explored the likelihood of resistance to DEG phenotypes arising, and how this risk may be reduced. The review is intended for a general audience and mathematical details are kept to a minimum.
Impact of mosquito gene drive on malaria elimination in a computational model with explicit spatial and temporal dynamics
4039Eckhoff, PAW, E. A.; Godfray, H. C. J.; Burt, A., Proceedings of the National Academy of Sciences of the United States of America, 114:e255-e264. 2017-01-17 00:00:00.
The renewed effort to eliminate malaria and permanently remove its tremendous burden highlights questions of what combination of tools would be sufficient in various settings and what new tools need to be developed. Gene drive mosquitoes constitute a promising set of tools, with multiple different possible approaches including population replacement with introduced genes limiting malaria transmission, driving-Y chromosomes to collapse a mosquito population, and gene drive disrupting a fertility gene and thereby achieving population suppression or collapse. Each of these approaches has had recent success and advances under laboratory conditions, raising the urgency for understanding how each could be deployed in the real world and the potential impacts of each. New analyses are needed as existing models of gene drive primarily focus on nonseasonal or nonspatial dynamics. We use a mechanistic, spatially explicit, stochastic, individual-based mathematical model to simulate each gene drive approach in a variety of sub-Saharan African settings. Each approach exhibits a broad region of gene construct parameter space with successful elimination of malaria transmission due to the targeted vector species. The introduction of realistic seasonality in vector population dynamics facilitates gene drive success compared with nonseasonal analyses. Spatial simulations illustrate constraints on release timing, frequency, and spatial density in the most challenging settings for construct success. Within its parameter space for success, each gene drive approach provides a tool for malaria elimination unlike anything presently available. Provided potential barriers to success are surmounted, each achieves high efficacy at reducing transmission potential and lower delivery requirements in logistically challenged settings.
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.
Requirements for Driving Antipathogen Effector Genes into Populations of Disease Vectors by Homing
4031Beaghton, AH, Andrew; Nolan, Tony; Crisanti, Andrea; Godfray, H. Charles J.; Burt, Austin, Genetics, 205:1587-1596. 2017-01-09 00:00:00.
There is a need for new interventions against the ongoing burden of vector-borne diseases such as malaria and dengue. One suggestion has been to develop genes encoding effector molecules that block parasite development within the vector, and then use the nuclease-based homing reaction as a form of gene drive to spread those genes through target populations. If the effector gene reduces the fitness of the mosquito and does not contribute to the drive, then loss-of-function mutations in the effector will eventually replace functional copies, but protection may nonetheless persist sufficiently long to provide a public health benefit. Here, we present a quantitative model allowing one to predict the duration of protection as a function of the probabilities of different molecular processes during the homing reaction, various fitness effects, and the efficacy of the effector in blocking transmission. Factors that increase the duration of protection include reducing the frequency of pre-existing resistant alleles, the probability of nonrecombinational DNA repair, the probability of homing-associated loss of the effector, the fitness costs of the nuclease and effector, and the completeness of parasite blocking. For target species that extend over an area much larger than the typical dispersal distance, the duration of protection is expected to be highest at the release site, and decrease away from there, eventually falling to zero, as effector-less drive constructs replace effector-containing ones. We also model an alternative strategy of using the nuclease to target an essential gene, and then linking the effector to a sequence that restores the essential function and is resistant to the nuclease. Depending upon parameter values, this approach can prolong the duration of protection. Our models highlight the key design criteria needed to achieve a desired level of public health benefit.%U http://www.genetics.org/content/genetics/205/4/1587.full.pdf
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.
Cheating evolution: engineering gene drives to manipulate the fate of wild populations
4083Champer, JB, A.; Akbari, O. S., Nature Reviews Genetics, 17:146-159. 2016-01-21 00:00:00.
Engineered gene drives - the process of stimulating the biased inheritance of specific genes - have the potential to enable the spread of desirable genes throughout wild populations or to suppress harmful species, and may be particularly useful for the control of vector-borne diseases such as malaria. Although several types of selfish genetic elements exist in nature, few have been successfully engineered in the laboratory thus far. With the discovery of RNA-guided CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated 9) nucleases, which can be utilized to create, streamline and improve synthetic gene drives, this is rapidly changing. Here, we discuss the different types of engineered gene drives and their potential applications, as well as current policies regarding the safety and regulation of gene drives for the manipulation of wild populations.
Gene drive through a landscape: Reaction-diffusion models of population suppression and elimination by a sex ratio distorter
4081Beaghton, AB, P. J.; Burt, A., Theoretical Population Biology, 108:51-69. 2016-01-19 00:00:00.
Some genes or gene complexes are transmitted from parents to offsprihg at a greater-than-Mendelian rate, and can spread and persist in populations even if they cause some harm to the individuals carrying them. Such genes may be useful for controlling populations or species that are harmful. Driving-Y chromosomes may be particularly potent in this regard, as they produce a male-biased sex ratio that, if sufficiently extreme, can lead to population elimination. To better understand the potential of such genes to spread over a landscape, we have developed a series of reaction-diffusion models of a driving-Y chromosome in 1-D and radially-symmetric 2-D unbounded domains. The wild-type system at carrying capacity is found to be unstable to the introduction of driving-Y males for all models investigated. Numerical solutions exhibit travelling wave pulses and fronts, and analytical and semi-analytical solutions for the asymptotic wave speed under bounded initial conditions are derived. The driving-Y male invades the wild-type equilibrium state at the front of the wave and completely replaces the wild-type males, leaving behind, at the tail of the wave, a reduced- or zero-population state of females and driving-Y males only. In our simplest model of a population with one life stage and density-dependent mortality, wave speed depends on the strength of drive and the diffusion rate of Y-drive males, and is independent of the population dynamic consequences (suppression or elimination). Incorporating an immobile juvenile stage of fixed duration into the model reduces wave speed approximately in proportion to the relative time spent as a juvenile. If females mate just once in their life, storing sperm for subsequent reproduction, then wave speed depends on the movement of mated females as well as Y-drive males, and may be faster or slower than in the multiple-mating model, depending on the relative duration of juvenile and adult life stages. Numerical solutions are shown for parameter values that may in part be representative for Anopheles gambiae, the primary vector of malaria in sub-Saharan Africa. (C) 2015 The Authors. Published by Elsevier Inc.
What is a Gene Drive?
4715STAT, 2015-12-11 00:00:00.
This video produced by STAT, an e-news site focusing on health and medicine (https://www.statnews.com/). This video simply illustrates what geneticists mean by gene drive, and how homing-based gene drive work. (Note: it does not indicate that there are other mechanisms of gene drive.)
CRISPR-Cas9: Safeguarding Gene Drives
5501Harvard University, 2015-11-16 18:44:48.
In this animation, learn how effective safeguarding mechanisms developed at the Wyss Institute and Harvard Medical School can be applied to ensure gene drive research is done responsibly in the laboratory. These safeguards enable responsible scientific investigation into how gene drives could one day be leveraged for the greater good of human health, agriculture, and the environment.
A synthetic sex ratio distortion system for the control of the human malaria mosquito
4140Galizi, RD, L. A.; Menichelli, M.; Bernardini, F.; Deredec, A.; Burt, A.; Stoddard, B. L.; Windbichler, N.; Crisanti, A., Nature Communications, 5:3977. 2014-01-18 00:00:00.
It has been theorized that inducing extreme reproductive sex ratios could be a method to suppress or eliminate pest populations. Limited knowledge about the genetic makeup and mode of action of naturally occurring sex distorters and the prevalence of co-evolving suppressors has hampered their use for control. Here we generate a synthetic sex distortion system by exploiting the specificity of the homing endonuclease I-PpoI, which is able to selectively cleave ribosomal gene sequences of the malaria vector Anopheles gambiae that are located exclusively on the mosquito's X chromosome. We combine structure-based protein engineering and molecular genetics to restrict the activity of the potentially toxic endonuclease to spermatogenesis. Shredding of the paternal X chromosome prevents it from being transmitted to the next generation, resulting in fully fertile mosquito strains that produce >95% male offspring. We demonstrate that distorter male mosquitoes can efficiently suppress caged wild-type mosquito populations, providing the foundation for a new class of genetic vector control strategies.
Modelling the spatial spread of a homing endonuclease gene in a mosquito population
4177North, AB, A.; Godfray, H. C. J., Journal of Applied Ecology, 50:1216-1225. 2013-01-15 00:00:00.
Homing endonuclease genes (HEGs) exist naturally in many single-celled organisms and can show extremely strong genetic drive allowing them to spread through populations into which they are introduced. They are being investigated as tools to manipulate the populations of important vectors of human disease, in particular the mosquitoes that transmit malaria. Before HEGs can be deployed, it is important to study their spatial spread in order to design efficient release strategies. A spatially explicit model is developed to study the spread of a HEG through a landscape whose structure is defined by the distribution of mosquito breeding and feeding sites. The model is motivated by the biology of the major vectors of malaria in Africa. The conditions for spread, fixation and loss of two major types of HEG are explored in different landscapes. In landscapes where mosquito resources are abundant, the conditions for spread are well approximated by a mean-field model. Where a HEG imposes a genetic load, it can cause population extinction, though spatial models more often predict population suppression. In certain types of landscapes where mosquito resources are rare, an introduced HEG may be prevented from moving between local mosquito populations and so a simple release strategy is unlikely to be effective, yet if the HEG succeeds in spreading population extinction is a feasible outcome. Increasing the number of release sites at the expense of releasing fewer mosquitoes per site reduces the probability that a HEG will fail.Synthesis and applications. The model presented asks for the first time how the spatial structure of mosquito populations will influence the effectiveness of a technology that is being rapidly developed for vector control. If homing endonuclease genes (HEGs) are to be used in this way, we have qualified the importance of accounting for landscape characteristics in both the execution and the expectation of their application. The next stage is to use the model to study the spread of HEGs through real landscapes where releases may take place, something that will be facilitated by the results of the present study. The model presented asks for the first time how the spatial structure of mosquito populations will influence the effectiveness of a technology that is being rapidly developed for vector control. If homing endonuclease genes (HEGs) are to be used in this way, we have qualified the importance of accounting for landscape characteristics in both the execution and the expectation of their application. The next stage is to use the model to study the spread of HEGs through real landscapes where releases may take place, something that will be facilitated by the results of the present study.
The design and in vivo evaluation of engineered I-OnuI-based enzymes for HEG gene drive
4169Chan, YST, R.; Jarjour, J.; Huen, D. S.; Stoddard, B. L.; Russell, S., PLOS One, 8:e74254. 2013-01-07 00:00:00.
The homing endonuclease gene (HEG) drive system, a promising genetic approach for controlling arthropod populations, utilises engineered nucleases to spread deleterious mutations that inactivate individual genes throughout a target population. Previous work with a naturally occurring LAGLIDADG homing endonuclease (I-SceI) demonstrated its feasibility in both Drosophila and Anopheles. Here we report on the next stage of this strategy: the redesign of HEGs with customized specificity in order to drive HEG-induced 'homing' in vivo via break-induced homologous recombination. Variants targeting a sequence within the Anopheles AGAP004734 gene were created from the recently characterized I-OnuI endonuclease, and tested for cleavage activity and frequency of homing using a model Drosophila HEG drive system. We observed cleavage and homing at an integrated reporter for all endonuclease variants tested, demonstrating for the first time that engineered HEGs can cleave their target site in insect germline cells, promoting targeted mutagenesis and homing. However, in comparison to our previously reported work with I-SceI, the engineered I-OnuI variants mediated homing with a reduced frequency, suggesting that site-specific cleavage activity is insufficient by itself to ensure efficient homing. Taken together, our experiments take a further step towards the development of a viable HEG-based population control strategy for insects.
Optimising homing endonuclease gene drive performance in a semi-refractory species: The Drosophila melanogaster experience
4168Chan, YSH, D. S.; Glauert, R.; Whiteway, E.; Russell, S., PLOS One, 8:e54130. 2013-01-06 00:00:00.
Homing endonuclease gene (HEG) drive is a promising insect population control technique that employs meganucleases to impair the fitness of pest populations. Our previous studies showed that HEG drive was more difficult to achieve in Drosophila melanogaster than Anopheles gambiae and we therefore investigated ways of improving homing performance in Drosophila. We show that homing in Drosophila responds to increased expression of HEGs specifically during the spermatogonia stage and this could be achieved through improved construct design. We found that 39-UTR choice was important to maximise expression levels, with HEG activity increasing as we employed Hsp70, SV40, vasa and beta Tub56D derived UTRs. We also searched for spermatogonium-specific promoters and found that the Rcd-1r promoter was able to drive specific expression at this stage. Since Rcd-1 is a regulator of differentiation in other species, it suggests that Rcd-1r may serve a similar role during spermatogonial differentiation in Drosophila. Contrary to expectations, a fragment containing the entire region between the TBPH gene and the bgcn translational start drove strong HEG expression only during late spermatogenesis rather than in the germline stem cells and spermatogonia as expected. We also observed that the fraction of targets undergoing homing was temperature-sensitive, falling nearly four-fold when the temperature was lowered to 18 degrees C. Taken together, this study demonstrates how a few simple measures can lead to substantial improvements in the HEG-based gene drive strategy and reinforce the idea that the HEG approach may be widely applicable to a variety of insect control programs.
Germline excision of transgenes in Aedes aegypti by homing endonucleases
4165Aryan, AA, M. A. E.; Myles, K. M.; Adelman, Z. N., Scientific Reports, 3:1603. 2013-01-03 00:00:00.
Aedes (Ae.) aegypti is the primary vector for dengue viruses (serotypes1-4) and chikungunya virus. Homing endonucleases (HEs) are ancient selfish elements that catalyze double-stranded DNA breaks (DSB) in a highly specific manner. In this report, we show that the HEs Y2-I-AniI, I-CreI and I-SceI are all capable of catalyzing the excision of genomic segments from the Ae. aegypti genome in a heritable manner. Y2-I-AniI demonstrated the highest efficiency at two independent genomic targets, with 20-40% of Y2-I-AniI-treated individuals producing offspring that had lost the target transgene. HE-induced DSBs were found to be repaired via the single-strand annealing (SSA) and non-homologous end-joining (NHEJ) pathways in a manner dependent on the availability of direct repeat sequences in the transgene. These results support the development of HE-based gene editing and gene drive strategies in Ae. aegypti, and confirm the utility of HEs in the manipulation and modification of transgenes in this important vector.
Requirements for effective malaria control with homing endonuclease genes
4203Deredec, AG, H. C. J.; Burt, A., Proceedings of the National Academy of Sciences of the United States of America, 108:e874-e880. 2011-01-21 00:00:00.
Malaria continues to impose a substantial burden on human health. We have previously proposed that biological approaches to control the mosquito vector of disease could be developed using homing endonuclease genes (HEGs), a class of selfish or parasitic gene that exists naturally in many microbes. Recent lab studies have demonstrated that HEGs can function in mosquitoes. We constructed and analyzed a model of mosquito population genetics and malaria epidemiology to determine how well HEGs need to function in order to have a significant effect on the burden of disease. Our model, combined with currently available data, indicates that populations of Anopheles gambiae could be eliminated by releasing 2-3 HEGs targeting female fertility genes, or a driving-Y chromosome that is transmitted to 75-96% of progeny. Combinations of fertility-targeting HEGs and Y drive may also be effective. It is possible to eliminate the disease without eliminating the vector, but the parameter space producing this outcome appears to be small. HEGs causing a quantitative reduction in adult survival can be more effective than those targeting female fertility, but the selection coefficients that need to be imposed are still large, unless many HEGs are to be released. Simulations show that HEG-based strategies can be effective over socially relevant time frames. Important limiting assumptions of the models are that there is only a single vector species, and we model a homogeneous population, not a landscape. Nevertheless, we conclude that HEG-based approaches could have a transformational effect on malaria control efforts.
Insect population control by homing endonuclease-based gene drive: An evaluation in Drosophila melanogaster
4201Chan, YSN, D. A.; Huen, D. S.; Russell, S., Genetics, 188:33-44. 2011-01-19 00:00:00.
Insects play a major role as vectors of human disease as well as causing significant agricultural losses. Harnessing the activity of customized homing endonuclease genes (HEGs) has been proposed as a method for spreading deleterious mutations through populations with a view to controlling disease vectors. Here, we demonstrate the feasibility of this method in Drosophila melanogaster, utilizing the well-characterized HEG, I-SceI. In particular, we show that high rates of homing can be achieved within spermatogonia and in the female germline. We show that homed constructs continue to exhibit HEG activity in the subsequent generation and that the ectopic homing events required for initiating the strategy occur at an acceptable rate. We conclude that the requirements for successful deployment of a HEG-based gene drive strategy can be satisfied in a model dipteran and that there is a reasonable prospect of the method working in other dipterans. In characterizing the system we measured repair outcomes at the spermatogonial, spermatocyte, and spermatid stages of spermatogenesis. We show that homologous recombination is restricted to spermatogonia and that it immediately ceases when they become primary spermatocytes, indicating that the choice of DNA repair pathway in the Drosophila testis can switch abruptly during differentiation.
A synthetic homing endonuclease-based gene drive system in the human malaria mosquito
4211Windbichler, NM, M.; Papathanos, P. A.; Thyme, S. B.; Li, H.; Ulge, U. Y.; Hovde, B. T.; Baker, D.; Monnat, R. J.; Burt, A.; Crisanti, A., Nature, 473:212-215. 2011-01-09 00:00:00.
Genetic methods of manipulating or eradicating disease vector populations have long been discussed as an attractive alternative to existing control measures because of their potential advantages in terms of effectiveness and species specificity(1-3). The development of genetically engineered malaria-resistant mosquitoes has shown, as a proof of principle, the possibility of targeting the mosquito's ability to serve as a disease vector(4-7). The translation of these achievements into control measures requires an effective technology to spread a genetic modification from laboratory mosquitoes to field populations(8). We have suggested previously that homing endonuclease genes (HEGs), a class of simple selfish genetic elements, could be exploited for this purpose(9). Here we demonstrate that a synthetic genetic element, consisting of mosquito regulatory regions(10) and the homing endonuclease gene I-SceI(11-13), can substantially increase its transmission to the progeny in transgenic mosquitoes of the human malaria vector Anopheles gambiae. We show that the I-SceI element is able to invade receptive mosquito cage populations rapidly, validating mathematical models for the transmission dynamics of HEGs. Molecular analyses confirm that expression of I-SceI in the male germline induces high rates of site-specific chromosomal cleavage and gene conversion, which results in the gain of the I-SceI gene, and underlies the observed genetic drive. These findings demonstrate a new mechanism by which genetic control measures can be implemented. Our results also show in principle how sequence-specific genetic drive elements like HEGs could be used to take the step from the genetic engineering of individuals to the genetic engineering of populations.
The population genetics of using homing endonuclease genes in vector and pest management
4234Deredec, AB, A.; Godfray, H. C. J., Genetics, 179:2013-2026. 2008-01-12 00:00:00.
Homing endonuclease genes (HEGs) encode proteins that in the heterozygous state cause double- strand breaks in the homologous chromosome at the precise position opposite the HFG. If the double-strand break is repaired using the homologous chromosome, the HEG becomes homozygous, and this represents it Powerful genetic drive mechanism that might he used as it tool managing vector or pest populations. HEGs may be used to decrease population fitness to drive down population densities (possibly causing local extinction) or, in disease vectors, to knock out a gene required for pathogen transmission. The relative advantages of HEGs thia target viability or fecundity, that are active in one sex or both, and whose target. is expressed before or after homing are explored. The conditions under which escape mutants arise are also analyzed. A different strategy is to place HEGs on the Y chromosome that cause one, or more breaks on the X chromosome and so disrupt. sex ratio. This strategy can cause severe sex-ratio biases with efficiencies that depend on the details of sperm Competition and zygote mortality. This strategy is probably less susceptible to escape mutants, especially when multiple X shredders are used.
Targeting the X chromosome during spermatogenesis induces Y chromosome transmission ratio distortion and early dominant embryo lethality in Anopheles gambiae
4247Windbichler, NP, P. A.; Crisanti, A., PLOS Genetics, 4:1-9. 2008-01-05 00:00:00.
We have exploited the high selectivity of the homing endonuclease I-PpoI for the X-linked Anopheles gambiae 28S ribosomal genes to selectively target X chromosome carrying spermatozoa. Our data demonstrated that in heterozygous males, the expression of I-PpoI in the testes induced a strong bias toward Y chromosome-carrying spermatozoa. Notably, these male mosquitoes also induced complete early dominant embryo lethality in crosses with wild-type females. Morphological and molecular data indicated that all spermatozoa, irrespectively of the inheritance of the transgene, carried a substantial amount of I-PpoI protein that could attack the maternally inherited chromosome X of the embryo. Besides the obvious implications for implementing vector control measures, our data demonstrated the feasibility of generating synthetic sex distorters and revealed the intriguing possibility of manipulating maternally inherited genes using wild-type sperm cells carrying engineered endonucleases.
Homing endonuclease mediated gene targeting in Anopheles gambiae cells and embryos
4259Windbichler, NP, P. A.; Catteruccia, F.; Ranson, H.; Burt, A.; Crisanti, A., Nucleic Acids Research, 35:5922-5933. 2007-01-17 00:00:00.
Homing endonuclease genes (HEGs) are selfish genetic elements that combine the capability to selectively disrupt specific gene sequences with the ability to rapidly spread from a few individuals to an entire population through homologous recombination repair events. Because of these properties, HEGs are regarded as promising candidates to transfer genetic modifications from engineered laboratory mosquitoes to wild-type populations including Anopheles gambiae the vector of human malaria. Here we show that I-SceI and I-PpoI homing endonucleases cleave their recognition sites with high efficiency inA. gambiae cells and embryos and we demonstrate HEG-induced homologous and non-homologous repair events in a variety of functional assays. We also propose a gene drive system for mosquitoes that is based on our finding that I-PpoI cuts genomic rDNA located on the X chromosome in A. gambiae, which could be used to selectively incapacitate X-carrying spermatozoa thereby imposing a severe male-biased sex ratio.
Degeneration and domestication of a selfish gene in yeast: Molecular evolution versus site-directed mutagenesis
4278Koufopanou, VB, A., Molecular Biology and Evolution, 22:1535-1538. 2005-01-16 00:00:00.
VDE is a homing endonuclease gene in yeasts with an unusual evolutionary history including horizontal transmission, degeneration, and domestication into the mating-type switching locus HO. We investigate here the effects of these features on its molecular evolution. In addition, we correlate rates of evolution with results from site-directed mutagenesis studies. Functional elements have, lower rates of evolution than degenerate ones and higher conservation at functionally important sites. However, functionally important and unimportant sites are equally likely to have been involved in the evolution of new function during the domestication of VDE into HO. The domestication event also indicates that VDE has been lost in some species and that VDE has been present in yeasts for more than 50 Myr.
Evolution of divergent DNA recognition specificities in VDE homing endonucleases from two yeast species
4299Posey, KLK, V.; Burt, A.; Gimble, F. S., Nucleic Acids Research, 32:3947-3956. 2004-01-17 00:00:00.
Homing endonuclease genes (HEGs) are mobile DNA elements that are thought to confer no benefit to their host. They encode site-specific DNA endonucleases that perpetuate the element within a species population by homing and disseminate it between species by horizontal transfer. Several yeast species contain the VMA1 HEG that encodes the intein-associated VMA1-derived endonuclease (VIDE). The evolutionary state of VDEs from 12 species was assessed by assaying their endonuclease activities. Only two enzymes are active, PI-ZbaI from Zygosaccharomyces bailii and PI-ScaI from Saccharomyces cariocanus. PI-ZbaI cleaves the Z-bailii recognition sequence significantly faster than the Saccharomyces cerevisiae site, which differs at six nucleotide positions. A mutational analysis indicates that PI-ZbaI cleaves the S.cerevisiae substrate poorly due to the absence of a contact that is analogous to one made in PI-SceI between Gln-55 and nucleotides +9/+10. PI-ZbaI cleaves the Z.bailii substrate primarily due to a single base-pair substitution (A/T+5 --> T/A(+5)). Structural modeling of the PI-ZbaI/DNA complex suggests that Arg-331, which is absent in PI-SceI, contacts T/A(+5), and the reduced activity observed in a PI-ZbaI R331 A mutant provides evidence for this interaction. These data illustrate that homing endonucleases evolve altered specificity as they adapt to recognize alternative target sites.
Homing endonuclease genes: the rise and fall and rise again of a selfish element
4286Burt, AK, V., Current Opinion in Genetics & Development, 14:609-615. 2004-01-04 00:00:00.
Homing endonuclease genes (HEGs) are selfish genetic elements that spread by first cleaving chromosomes that do not contain them and then getting copied across to the broken chromosome as a byproduct of the repair process. The success of this strategy will depend on the opportunities for homing - in other words, the frequency with which HEG(+) and HEG(-) chromosomes come into contact - which varies widely among host taxa. HEGs are also unusual in that the selection pressure for endonuclease function disappears if they become fixed in a population, which makes them susceptible to degeneration and imposes a need for regular horizontal transmission between species. HEGs will be selected to reduce the harm done to the host organism, and this is expected to influence the evolution of their sequence specificity and maturase functions. HEGs may also be domesticated by their hosts, and are currently being put to human uses.
Site-specific selfish genes as tools for the control and genetic engineering of natural populations
4306Burt, A, Proceedings of the Royal Society B-Biological Sciences, 270:921-928. 2003-01-04 00:00:00.
Site-specific selfish genes exploit host functions to copy themselves into a defined target DNA sequence, and include homing endonuclease genes, group II introns and some LINE-like transposable elements. If such genes can be engineered to target new host sequences, then they can be used to manipulate natural populations, even if the number of individuals released is a small fraction of the entire population. For example, a genetic load sufficient to eradicate a population can be imposed in fewer than 20 generations, if the target is an essential host gene, the knockout is recessive and the selfish gene has an appropriate promoter. There will be selection for resistance, but several strategies are available for reducing the likelihood of it evolving. These genes may also be used to genetically engineer natural populations, by means of population-wide gene knockouts, gene replacements and genetic transformations. By targeting sex-linked loci just prior to meiosis one may skew the population sex ratio, and by changing the promoter one may limit the spread of the gene to neighbouring populations. The proposed constructs are evolutionarily stable in the face of the mutations most likely to arise during their spread, and strategies are also available for reversing the manipulations.
Adaptation for horizontal transfer in a homing endonuclease
4323Koufopanou, VG, M. R.; Burt, A., Molecular Biology and Evolution, 19:239-246. 2002-01-21 00:00:00.
Selfish genes of no function other than self-propagation are susceptible to degeneration if they become fixed in a population. and regular transfer to new species may be the only means for their long-term persistence. To test this idea we surveyed 24 species of yeast for VDE, a nuclear. intein-associated homing endonuclease gene (HEG) originally discovered in Saccharomyces cerevisiae. Phylogenetic analyses show that horizontal transmission has been a regular occurrence in its evolutionary history. Moreover, VDE appears to be specifically adapted for horizontal transmission. Its 31-bp recognition sequence is an unusually well-conserved region in an unusually well-conserved gene. In addition, the nine nucleotide sites most critical for homing are also unusually well conserved. Such adaptation for horizontal transmission presumably arose as a consequence of selection, both among HEGs at different locations in the genome and among variant,,, at the same location. The frequency of horizontal transmission must therefore be a key feature constraining the distribution and abundance of these genes.
Outcrossed sex allows a selfish gene to invade yeast populations
4329Goddard, MRG, D.; Burt, A., Proceedings of the Royal Society B-Biological Sciences, 268:2537-2542. 2001-01-07 00:00:00.
Homing endonuclease genes (HEGs) in eukaryotes are optional genes that have no obvious effect on host phenotype except for causing chromosomes not containing a cop), of the gene to be cut, thus causing them to be inherited at a greater than Mendelian rate via gene conversion. These genes are therefore expected to increase in frequency in outcrossed populations, but not in obligately selfed populations. In order to test this idea, we compared the dynamics of the VDE HEG in six replicate outcrossed and inbred populations of yeast (Saccharomyces cerevisiae). VDE increased in frequency from 0.21 to 0.55 in four outcrossed generations, but showed no change in frequency in the inbred populations. The absence of change in the inbred populations indicates that any effect of VDE on mitotic replication rates is less than 1%. The data from the outcrossed populations best fit a model in which 82% of individuals are derived from outcrossing and VDE is inherited by 74% of the meiotic products from heterozygotes (as compared with 50% for Mendelian genes), These results empirically demonstrate how a host mating system plays a key role in determining the population dynamics of a selfish gene.
Recurrent invasion and extinction of a selfish gene
4353Goddard, MRB, A., Proceedings of the National Academy of Sciences of the United States of America, 96:13880-13885. 1999-01-11 00:00:00.
Homing endonuclease genes show super-Mendelian inheritance, which allows them to spread in populations even when they are of no benefit to the host organism. To test the idea that regular horizontal transmission is necessary for the long-term persistence of these genes, we surveyed 20 species of yeasts for the omega-homing endonuclease gene and associated group I intron. The status of omega could be categorized into three states (functional, nonfunctional, or absent), and status was not clustered on the host phylogeny. Moreover, the phylogeny of w differed significantly from that of the host, strong evidence of horizontal transmission. Further analyses indicate that horizontal transmission is more common than transposition, and that it occurs preferentially between closely related species. Parsimony analysis and coalescent theory suggest that there have been 15 horizontal transmission events in the ancestry of our yeast species, through simulations indicate that this value is probably an underestimate. Overall, the data support a cyclical model of invasion, degeneration, and loss, followed by reinvasion, and each of these transitions is estimated to occur about once every 2 million years. The data are thus consistent with the idea that frequent horizontal transmission is necessary for the long-term persistence of homing endonuclease genes, and further, that this requirement limits these genes to organisms with easily accessible germ lines. The data also show that mitochondrial DNA sequences are transferred intact between yeast species; if other genes do not show such high levels of horizontal transmission, it would be due to lack of selection, rather than lack of opportunity.
Genetics-driving genes and chromosomes
4463Charlesworth, B, Nature, 332:394-395. 1988-01-21 00:00:00.
Thereare several genetic and chromosomal systems in which Mendel's first law - the equal probability of transmission of maternal and paternal alternative alleles or homologues - is violated. This phenomenon was named 'meiotic drive' in 1957 by Sandler and Novitski, who drew attention to the fact that it operates as an evolutionary force which can cause an increase in the population frequency of the allele or chromosome which is favoured in transmission, even if it confers a disadvantage on its carriers in terms of fitness at the level of the individual
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