Scholarly Literature

This is a database of scholarly literature that concentrates currently on natural and engineered selfish genetic elements (gene drives).  The latest are shown here.
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A multiplexed, confinable CRISPR/Cas9 gene drive propagates in caged Aedes aegypti populations

23429
M. A. E. Anderson, E. Gonzalez, M. P. Edgington, J. X. D. Ang, D.-K. Purusothaman, L. Shackleford, K. Nevard, S. A. N. Verkuijl, T. Harvey-Samuel, P. T. Leftwich, K. Esvelt and L. Alphey,  bioRxiv,  2022.08.12.503466. 2022-08-12 07:19:58.
Here, we test the regulatory sequences from the Ae. aegypti benign gonial cell neoplasm (bgcn) homolog to express Cas9 in the germline to find an expression timing more conducive to homing. We also created a separate multiplexing (targeting multiple different sites within the target gene) sgRNA-expressing homing cassette inserted into the Ae. aegypti kynurenine 3-monooxygenase (kmo) gene to limit the consequences of resistance alleles. This creates a ‘split’ gene drive such that one part does not drive, allowing control over geographic spread and temporal persistence. When combined, these two elements provide highly effective germline cutting at the kmo locus and act as a gene drive. Our target genetic element was driven through a cage trial population such that carrier frequency of the element increased from 50% to up to 89% of the population despite significant fitness costs to kmo insertions. Deep sequencing suggests that the multiplexing design could mitigate resistance allele formation in our gene drive system.Significance statement Mosquito-borne diseases affect millions of people worldwide, with the yellow fever mosquito (Aedes aegypti) being the principal vector of many viral diseases. Effective measures for controlling this mosquito are sorely needed. Gene drive systems have arisen as a potential tool for mosquito control due to their ability of biasing inheritance of a trait into a target population. Here, we assess a split gene drive, based on CRISPR/Cas9 endonuclease technology driving a target element into the mosquito population. Evaluated over successive generations in a replicated cage trial, the drive successfully biased its inheritance, increasing in frequency from 50% to up to 89%. Our results are encouraging for the potential use of this type of contained gene drive system for mosquito control in endemic areas.Competing Interest StatementThe authors have declared no competing interest.

Harnessing Wolbachia cytoplasmic incompatibility alleles for confined gene drive: a modeling study

23424
J. Li and J. Champer,  bioRxiv,  2022.08.09.503337. 2022-08-11 07:25:47.
By using both mathematical and simulation models, we found that a drive containing CifA and CifB together create a confined drive with a moderate to high introduction threshold. When introduced separately, they act as a self-limiting drive. We observed that the performance of these drives is substantially influenced by various ecological parameters and drive characteristics. Extending our models to continuous space, we found that the drive individual release distribution has a critical impact on drive persistence.

Aedes aegypti and Ae. albopictus microbiome/virome: new strategies for controlling arboviral transmission?

23413
M. Gómez, D. Martinez, M. Muñoz and J. D. Ramírez,  Parasites and Vectors,  15:287. 2022-08-09 12:21:12.
Aedes aegypti and Aedes albopictus are the main vectors of highly pathogenic viruses for humans, such as dengue (DENV), chikungunya (CHIKV), and Zika (ZIKV), which cause febrile, hemorrhagic, and neurological diseases and remain a major threat to global public health. The high ecological plasticity, opportunistic feeding patterns, and versatility in the use of urban and natural breeding sites of these vectors have favored their dispersal and adaptation in tropical, subtropical, and even temperate zones. Due to the lack of available treatments and vaccines, mosquito population control is the most effective way to prevent arboviral diseases. Resident microorganisms play a crucial role in host fitness by preventing or enhancing its vectorial ability to transmit viral pathogens. High-throughput sequencing and metagenomic analyses have advanced our understanding of the composition and functionality of the microbiota of Aedes spp. Interestingly, shotgun metagenomics studies have established that mosquito vectors harbor a highly conserved virome composed of insect-specific viruses (ISV). Although ISVs are not infectious to vertebrates, they can alter different phases of the arboviral cycle, interfering with transmission to the human host. Therefore, this review focuses on the description of Ae. aegypti and Ae. albopictus as vectors susceptible to infection by viral pathogens, highlighting the role of the microbiota-virome in vectorial competence and its potential in control strategies for new emerging and re-emerging arboviruses.

Reflection on the Challenges, Accomplishments, and New Frontiers of Gene Drives

23416
M. Melesse Vergara, J. Labbé and J. Tannous,  BioDesign Research,  2022:9853416. 2022-08-09 07:29:59.
Ongoing pest and disease outbreaks pose a serious threat to human, crop, and animal lives, emphasizing the need for constantgenetic discoveries that could serve as mitigation strategies. Gene drives are genetic engineering approaches discovered decadesago that may allow quick, super-Mendelian dissemination of genetic modifications in wild populations, offering hopes formedicine, agriculture, and ecology in combating diseases. Following itsfirst discovery, several naturally occurring selfishgenetic elements were identified and several gene drive mechanisms that could attain relatively high threshold populationreplacement have been proposed. This review provides a comprehensive overview of the recent advances in gene drive researchwith a particular emphasis on CRISPR-Cas gene drives, the technology that has revolutionized the process of genomeengineering. Herein, we discuss the benefits and caveats of this technology and place it within the context of natural genedrives discovered to date and various synthetic drives engineered. Later, we elaborate on the strategies for designing syntheticdrive systems to address resistance issues and prevent them from altering the entire wild populations. Lastly, we highlight themajor applications of synthetic CRISPR-based gene drives in different living organisms, including plants, animals, andmicroorganisms.

Sterility of Cydia pomonella by X ray irradiation as an alternative to gamma radiation for the sterile insect technique

23463
J.-H. Zhang, N. Li, H.-Y. Zhao, Y.-Q. Wang, X.-Q. Yang and K.-M. Wu,  Bulletin of Entomological Research,  2022-08-06 09:27:59.
The codling moth Cydia pomonella is a major pest of global significance impacting pome fruits and walnuts. It threatens the apple industry in the Loess Plateau and Bohai Bay in China. Sterile insect technique (SIT) could overcome the limitations set by environmentally compatible area-wide integrated pest management (AW-IPM) approaches such as mating disruption and attract-kill that are difficult to suppress in a high-density pest population, as well as the development of insecticide resistance. In this study, we investigated the effects of X-ray irradiation (183, 366, 549 Gy) on the fecundity and fertility of a laboratory strain of C. pomonella, using a newly developed irradiator, to evaluate the possibility of X-rays as a replacement for Cobalt60 (60Co-γ) and the expanded future role of this approach in codling moth control. Results show that the 8th-day is the optimal age for irradiation of male pupae. The fecundity decreased significantly as the dosage of radiation increased. The mating ratio and mating number were not influenced. However, treated females were sub-sterile at a radiation dose of 183 Gy (20.93%), and were almost 100% sterile at a radiation dose of 366 Gy or higher. Although exposure to a radiation dose of 366 Gy resulted in a significant reduction in the mating competitiveness of male moths, our radiation biology results suggest that this new generation of X-ray irradiator has potential applications in SIT programs for future codling moth control.

Studies on the fitness characteristics of wMel- and wAlbB-introgressed Aedes aegypti (Pud) lines in comparison with wMel- and wAlbB-transinfected Aedes aegypti (Aus) and wild-type Aedes aegypti (Pud) lines

23485
C. Sadanandane, K. Gunasekaran, D. Panneer, S. K. Subbarao, M. Rahi, B. Vijayakumar, V. Athithan, A. Sakthivel, S. Dinesh and P. Jambulingam,  Frontiers in Microbiology,  13:947857. 2022-08-05 07:02:55.
Wolbachia, an intracellular maternally transmitted endosymbiont, has been shown to interfere with the replication of dengue virus in Aedes aegypti mosquitoes. The Wolbachia-transinfected Ae. aegypti has been currently released in many countries to test its effectiveness in preventing the transmission of dengue virus. ICMR-Vector Control Research Centre in collaboration with World Mosquito Program Monash University, Australia, has generated two new Wolbachia-introgressed Ae. aegypti Puducherry (Pud) lines via backcrossing Ae. aegypti females of Australian (Aus) strains, infected with wMel and wAlbB Wolbachia with wild-type Ae. aegypti Puducherry (Pud) males. Wolbachia infections are known to induce a fitness cost and confer benefit on the host mosquito populations that will influence spread of the Wolbachia into native wild mosquito populations during the field release. Hence, the induced fitness cost or benefit/advantage in the two newly generated Ae. aegypti (Pud) lines was assessed in the laboratory in comparison with the wild-type Ae. aegypti (Pud) strain. In addition, maternal transmission (MT) efficiency, induced cytoplasmic incompatibility (CI), and insecticide resistance status of the two (Pud) lines were determined to assess the likely frequency of wMel and wAlbB infections in the native wild population after field invasion. The study shows that wMel and wAlbB infections did not induce any fitness cost on the two newly generated (Pud) lines. Rather, in terms of wing length, fecundity, egg hatch rate, and adult survival, the Wolbachia introgression conferred fitness benefits on the (Pud) lines compared to uninfected Wolbachia free wild Ae. aegypti population. wMel and wAlbB exhibited a high maternal transmission (99-100%) and induced nearly complete (98-100%) cytoplasmic incompatibility. Both the (Pud) lines were resistant to deltamethrin, malathion, DDT, and temephos, and the level of resistance was almost the same between the two lines as in the wild type. Overall, the stable association of wMel and wAlbB established with Ae. aegypti and the reproductive advantages of the (Pud) lines encourage a pilot release in the field for population replacement potential.

Wolbachia Dynamics in Mosquitoes with Incomplete CI and Imperfect Maternal Transmission by a DDE System

23393
Y. Su, B. Zheng and X. Zou,  Bulletin of Mathematical Biology,  84:95. 2022-08-01 08:09:11.
In this paper, we propose a delay differential equation model to describe the Wolbachia infection dynamics in mosquitoes in which the key factor of cytoplasmic incompactibility (CI) is incorporated in a more natural way than those in the literature. By analyzing the dynamics of the model, we are able to obtain some information on the impact of four important parameters: the competition capabilities of the wild mosquitoes and infected mosquitoes, the maternal transmission level and the CI level. The analytic results show that there are ranges of parameters that support competition exclusion principle, and there are also ranges of parameters that allow co-persistence for both wild and infected mosquitoes. These ranges account for the scenarios of failure of invasion, invasion and suppressing the wild mosquitoes, and invasion and replacing the wild mosquitoes. We also discuss some possible future problems both in mathematics and in modeling.

A theory of resistance to multiplexed gene drive demonstrates the significant role of weakly deleterious natural genetic variation

23362
B. 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.

Robust control strategy by the Sterile Insect Technique for reducing epidemiological risk in presence of vector migration

23331
P.-A. Bliman and Y. Dumont,  Mathematical Biosciences,  350:108856. 2022-07-28 08:36:35.
The Sterile Insect Technique (SIT) is a promising technique to control mosquitoes, vectors of diseases, like dengue, chikungunya or Zika. However, its application in the field is not easy, and its success hinges upon several constraints, one of them being that the treated area must be sufficiently isolated to limit migration or re-invasion by mosquitoes from the outside. In this manuscript we study the impact of males and (fertile) females migration on SIT. We show that a critical release rate for sterile males exists for every migration level, in the context of continuous or periodic releases. In particular, when (fertile) females migration is sufficiently low, then SIT can be conducted successfully using either open-loop control or closed-loop control (or a combination of both methods) when regular measurements of the wild population are completed. Numerical simulations to illustrate our theoretical results are presented and discussed. Finally, we derive a threshold value for the females migration rate, when viruses are circulating, under which it is possible to lower the epidemiological risk in the treated area, according to the size of the human population.

Wolbachia wPip Blocks Zika Virus Transovarial Transmission in Aedes albopictus

23333
Y. Guo, J. Guo, Y. Li, X. Zheng and Y. Wu,  Microbiol Spectrum,  e0263321. 2022-07-27 08:36:55.
Area-wide application of Wolbachia to suppress mosquito populations and their transmitted viruses has achieved success in multiple countries. However, the mass release of Wolbachia-infected male mosquitoes involves a potential risk of accidentally releasing fertile females. In this study, we employed ovarian cells of the Ae. albopictus GUA, HC, and GT lines, which exhibit key traits, and compared them to better understand how Wolbachia inhibits ZIKV transovarial transmission. Our results showed an almost complete blockade of ZIKV transmission in HC female mosquitoes. Wolbachia in natively infected GUA mosquitoes negative affected ZIKV, and this interference was shown by slightly lower loads than those in HC mosquitoes. Overall, our work helps show how Wolbachia blocks ZIKV expansion and maintenance in the ovaries of Ae. albopictus and aids in understanding Wolbachia-ZIKV interactions in mosquitoes.

Developing Wolbachia-based disease interventions for an extreme environment

23309
P. A. Ross, S. Elfekih, S. Collier, M. J. Klein, S. S. Lee, M. Dunn, S. Jackson, Y. Zhang, J. K. Axford, X. Gu, M. S. Nasar, P. N. Paradkar, E. A. Taoufik, F. M. Jiggins, A. M. Almalik, M. B. Al-Fageeh and A. A. Hoffmann,  bioRxiv,  2022.07.26.501527. 2022-07-27 07:59:12.
Aedes aegypti mosquitoes carrying self-spreading, virus-blocking Wolbachia bacteria are being deployed to suppress dengue transmission. However, there are challenges in applying this technology in extreme environments. We introduced two Wolbachia strains into Ae. aegypti from Saudi Arabia for a release program in the hot coastal city of Jeddah. Wolbachia reduced infection and dissemination of dengue virus (DENV2) in Saudi Arabian mosquitoes and showed complete maternal transmission and cytoplasmic incompatibility. Wolbachia reduced mosquito heat tolerance and egg viability, with the Wolbachia strains showing differential thermal stability. Wolbachia effects were similar across mosquito genetic backgrounds but we found evidence of local adaptation, with Saudi Arabian mosquitoes having lower egg viability but higher adult desiccation tolerance than Australian mosquitoes. Genetic background effects will influence Wolbachia invasion dynamics, reinforcing the need to use local genotypes for mosquito release programs, particularly in extreme environments like Jeddah. Our comprehensive characterization of Wolbachia strains provides a foundation for Wolbachia-based disease interventions in harsh climates.Competing Interest StatementThe authors have declared no competing interest.

Population replacement gene drive characteristics for malaria elimination in a range of seasonal transmission settings: a modelling study

23307
S. Leung, N. Windbichler, E. A. Wenger, C. A. Bever and P. Selvaraj,  Malaria Journal,  21:226. 2022-07-26 07:49:07.
BACKGROUND: Gene drives are a genetic engineering method where a suite of genes is inherited at higher than Mendelian rates and has been proposed as a promising new vector control strategy to reinvigorate the fight against malaria in sub-Saharan Africa. METHODS: Using an agent-based model of malaria transmission with vector genetics, the impacts of releasing population-replacement gene drive mosquitoes on malaria transmission are examined and the population replacement gene drive system parameters required to achieve local elimination within a spatially-resolved, seasonal Sahelian setting are quantified. The performance of two different gene drive systems-"classic" and "integral"-are evaluated. Various transmission regimes (low, moderate, and high-corresponding to annual entomological inoculation rates of 10, 30, and 80 infectious bites per person) and other simultaneous interventions, including deployment of insecticide-treated nets (ITNs) and passive healthcare-seeking, are also simulated. RESULTS: Local elimination probabilities decreased with pre-existing population target site resistance frequency, increased with transmission-blocking effectiveness of the introduced antiparasitic gene and drive efficiency, and were context dependent with respect to fitness costs associated with the introduced gene. Of the four parameters, transmission-blocking effectiveness may be the most important to focus on for improvements to future gene drive strains because a single release of classic gene drive mosquitoes is likely to locally eliminate malaria in low to moderate transmission settings only when transmission-blocking effectiveness is very high (above ~ 80-90%). However, simultaneously deploying ITNs and releasing integral rather than classic gene drive mosquitoes significantly boosts elimination probabilities, such that elimination remains highly likely in low to moderate transmission regimes down to transmission-blocking effectiveness values as low as ~ 50% and in high transmission regimes with transmission-blocking effectiveness values above ~ 80-90%. CONCLUSION: A single release of currently achievable population replacement gene drive mosquitoes, in combination with traditional forms of vector control, can likely locally eliminate malaria in low to moderate transmission regimes within the Sahel. In a high transmission regime, higher levels of transmission-blocking effectiveness than are currently available may be required.

Non-Mendelian transmission of accessory chromosomes in fungi

23305
J. Komluski, E. H. Stukenbrock and M. Habig,  Chromosome Research,  2022-07-26 07:44:07.
Non-Mendelian transmission has been reported for various genetic elements, ranging from small transposons to entire chromosomes. One prime example of such a transmission pattern are B chromosomes in plants and animals. Accessory chromosomes in fungi are similar to B chromosomes in showing presence/absence polymorphism and being non-essential. How these chromosomes are transmitted during meiosis is however poorly understood-despite their often high impact on the fitness of the host. For several fungal organisms, a non-Mendelian transmission or a mechanistically unique meiotic drive of accessory chromosomes have been reported. In this review, we provide an overview of the possible mechanisms that can cause the non-Mendelian transmission or meiotic drives of fungal accessory chromosomes. We compare processes responsible for the non-Mendelian transmission of accessory chromosomes for different fungal eukaryotes and discuss the structural traits of fungal accessory chromosomes affecting their meiotic transmission. We conclude that research on fungal accessory chromosomes, due to their small size, ease of sequencing, and epigenetic profiling, can complement the study of B chromosomes in deciphering factors that influence and regulate the non-Mendelian transmission of entire chromosomes.

Lack of robust evidence for a Wolbachia infection in Anopheles gambiae from Burkina Faso

23300
S. P. Sawadogo, D. A. Kabore, E. B. Tibiri, A. Hughes, O. Gnankine, S. Quek, A. Diabaté, H. Ranson, G. L. Hughes and R. K. Dabiré,  Medical and Veterinary Entomology,  2022-07-25 07:31:44.
The endosymbiont Wolbachia can have major effects on the reproductive fitness, and vectorial capacity of host insects and may provide new avenues to control mosquito-borne pathogens. Anopheles gambiae s.l is the major vector of malaria in Africa but the use of Wolbachia in this species has been limited by challenges in establishing stable transinfected lines and uncertainty around native infections. High frequencies of infection of Wolbachia have been previously reported in An. gambiae collected from the Valle du Kou region of Burkina Faso in 2011 and 2014. Here, we re-evaluated the occurrence of Wolbachia in natural samples, collected from Valle du Kou over a 12-year time span, and in addition, expanded sampling to other sites in Burkina Faso. Our results showed that, in contrast to earlier reports, Wolbachia is present at an extremely low prevalence in natural population of An. gambiae. From 5341 samples analysed, only 29 were positive for Wolbachia by nested PCR representing 0.54% of prevalence. No positive samples were found with regular PCR. Phylogenetic analysis of 16S rRNA gene amplicons clustered across supergroup B, with some having similarity to sequences previously found in Anopheles from Burkina Faso. However, we cannot discount the possibility that the amplicon positive samples we detected were due to environmental contamination or were false positives. Regardless, the lack of a prominent native infection in An. gambiae s.l. is encouraging for applications utilizing Wolbachia transinfected mosquitoes for malaria control.

Operationalizing stakeholder engagement for gene drive research in malaria elimination in Africa-translating guidance into practice

23281
L. Pare Toe, B. Dicko, R. Linga, N. Barry, M. Drabo, N. Sykes and D. Thizy,  Malaria Journal,  21:225. 2022-07-23 10:16:04.
Gene drive mosquitoes are increasingly considered a potential transformational tool for vector control of malaria mosquitoes. As part of efforts to promote responsible research in this field, a number of guidance documents have been published by the World Health Organization, National Academies and expert groups. While virtually all recent guidance documents on gene drive research stress the importance of stakeholder engagement activities, no specific guidelines on implementing them have been established. Target Malaria, a not-for-profit research consortium developing a vector-control gene drive approach to eliminate malaria, has reflected on how its stakeholder engagement strategy translates engagement guidance documents into practice. The project analysed and addressed the tension between the context specificities and the international recommendations. The engagement strategy combines published recommendations for responsible gene drive research, information collected from the local context where the project operates and a set of principles guiding the choices made. This strategy was first developed during the early phases of the project's research, years ahead of any activities with gene drive mosquitoes in those countries of operations. These earlier activities, and their related engagement, allow the project to develop and adapt an engagement strategy appropriate for potential gene drive research in its field site countries. This paper offers a description of a stakeholder engagement strategy operationalization based on (1) adaptation to stakeholder preferences, (2) inclusiveness and (3) empowerment and accountability. The authors hope to offer concrete examples to support other projects with the development and implementation of their engagement strategies with particular attention to the co-development principle.

Attempts to use breeding approaches in Aedes aegypti to create lines with distinct and stable relative Wolbachia densities

23279
A. J. Mejia, L. Jimenez, H. L. C. Dutra, R. Perera and E. A. McGraw,  Heredity,  2022-07-22 10:12:25.
Wolbachia is an insect endosymbiont being used for biological control in the mosquito Aedes aegypti because it causes cytoplasmic incompatibility (CI) and limits viral replication of dengue, chikungunya, and Zika viruses. While the genetic mechanism of pathogen blocking (PB) is not fully understood, the strength of both CI and PB are positively correlated with Wolbachia densities in the host. Wolbachia densities are determined by a combination of Wolbachia strain and insect genotype, as well as interactions with the environment. We employed both artificial selection and inbreeding with the goal of creating lines of Ae. aegypti with heritable and distinct Wolbachia densities so that we might better dissect the mechanism underlying PB. We were unable to shift the mean relative Wolbachia density in Ae. aegypti lines by either strategy, with relative densities instead tending to cycle over a narrow range. In lieu of this, we used Wolbachia densities in mosquito legs as predictors of relative densities in the remaining individual’s carcass. Because we worked with outbred mosquitoes, our findings indicate either a lack of genetic variation in the mosquito for controlling relative density, natural selection against extreme densities, or a predominance of environmental factors affecting densities. Our study reveals that there are moderating forces acting on relative Wolbachia densities that may help to stabilize density phenotypes post field release. We also show a means to accurately bin vector carcasses into high and low categories for non-DNA omics-based studies of Wolbachia-mediated traits.

Comprehensive characterization of a transgene insertion in a highly repetitive, centromeric region of Anopheles mosquitoes

23273
M. Vitale, C. Leo, T. Courty, N. Kranjc, J. B. Connolly, G. Morselli, C. Bamikole, R. E. Haghighat-Khah, F. Bernardini and S. Fuchs,  Pathogens and Global Health,  2022-07-21 07:55:48.
The availability of the genomic sequence of the malaria mosquito Anopheles gambiae has in recent years sparked the development of transgenic technologies with the potential to be used as novel vector control tools. These technologies rely on genome editing that confer traits able to affect vectorial capacity. This can be achieved by either reducing the mosquito population or by making mosquitoes refractory to the parasite infection. For any genetically modified organism that is regarded for release, molecular characterization of the transgene and flanking sites are essential for their safety assessment and post-release monitoring. Despite great advancements, Whole-Genome Sequencing data are still subject to limitations due to the presence of repetitive and unannotated DNA sequences. Faced with this challenge, we describe a number of techniques that were used to identify the genomic location of a transgene in the male bias mosquito strain Ag(PMB)1 considered for potential field application. While the initial inverse PCR identified the most likely insertion site on Chromosome 3 R 36D, reassessment of the data showed a high repetitiveness in those sequences and multiple genomic locations as potential insertion sites of the transgene. Here we used a combination of DNA sequencing analysis and in-situ hybridization to clearly identify the integration of the transgene in a poorly annotated centromeric region of Chromosome 2 R 19D. This study emphasizes the need for accuracy in sequencing data for the genome of organisms of medical importance such as Anopheles mosquitoes and other tools available that can support genomic locations of transgenes.

Development of CRISPR/Cas9-Mediated Gene-Drive Construct Targeting the Phenotypic Gene in Plutella xylostella

23265
M. 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.

What can we learn from selfish loci that break Mendel’s law?

23349
S. E. Zanders,  PLOS Biology,  20:e3001700. 2022-07-19 07:31:24.
Mendel’s law of segregation provides a critical foundation for genetic inquiry It is not, however, without exceptions Historically, 2 such exceptions (sex chromosome linkage and chromosome missegregation in meiosis) were used by Drosophila geneticists to help demonstrate that genes are carried on chromosomes Looking forward, modern geneticists interested in understanding the mechanisms of heredity have much to learn from additional exceptions to Mendel’s law In particular, selfish genes that break Mendel’s law of segregation to gain a transmission advantage into the next generation are likely to be oversized contributors to shaping the process of sexual reproduction These selfish genes exploit reproduction such that a given selfish locus is transmitted to more than half of the offspring produced by an organism There are a variety of selfish DNAs, but here I will focus on transposable elements and drive loci as examples Transposable elements can generate novel copies of themselves using copy and paste or cut and paste mechanisms Importantly, transposable elements are selected to mobilize in the germline as that allows new copies to be passed on to subsequent generations Drive loci preferentially bias their own transmission such that a driver+ /driver− heterozygote will pass the driver+ allele to more than half of its viable progeny Drivers are diverse and can act during meiosis, gametogenesis, or post-fertilization Both transposable elements and drivers are found throughout eukaryotes, including humans

Isolation of rfk-2 (UV) , a mutation that blocks spore killing by Neurospora Spore killer-3

23382
A. Velazquez, E. Webber, D. O'Neil, T. Hammond and N. Rhoades,  MicroPublication Biology,  2022-07-17 07:16:34.
Neurospora Spore killer-3 ( Sk-3 ) is a selfish genetic element that kills spores to achieve gene drive.  Here, to help identify Sk-3’s killer, we performed a genetic screen for required for killing (rfk) mutations (see methods). The genetic screen uses Sk‑3 rskΔ × SkS crosses, which abort development before the production of viable ascospores (Hammond et al. 2012; Harvey et al. 2014). We isolated a few candidate rfk mutations with our genetic screen and chose the most promising candidate, rfk-2UV, for additional analysis. As demonstrated in Figure 1 (A and B), rfk-2UV disrupts spore killing and gene drive. To determine the approximate genomic location of rfk-2UV, we performed two sets of three-point crosses (Figure 1C). Recombination analysis of 213 offspring from the first set of crosses (rfk-2UV hphA × mus-52Δ) indicates that rfk-2UV islocated 2.8 cM from hphA and 15.0 cM from mus-52Δ (Figure 1D). For the second set of crosses (rfk-2UV hphB × mus-52Δ), recombination analysis of 186 offspring indicates that rfk-2UV is located 7.5 cM from hphB and 16.1 cM from mus-52Δ (Figure 1E).  The significance of this discovery with respect to Sk-3 evolution is discussed.

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