Transgenic expression of cif genes from Wolbachia strain wAlbB recapitulates cytoplasmic incompatibility in Aedes aegypti.

The endosymbiotic bacteria Wolbachia can invade insect populations by modifying host reproduction through cytoplasmic incompatibility (CI), an effect that results in embryonic lethality when Wolbachia-carrying males mate with Wolbachia-free females. Here we describe a transgenic system for recreating CI in the major arbovirus vector Aedes aegypti using CI factor (cif) genes from wAlbB, a Wolbachia strain currently being deployed to reduce dengue transmission. CI-like sterility is induced when cifA and cifB are co-expressed in testes; this sterility is rescued by maternal cifA expression, thereby reproducing the pattern of Wolbachia-induced CI. Expression of cifB alone is associated with extensive DNA damage and disrupted spermatogenesis. The strength of rescue by maternal cifA expression is dependent on the comparative levels of cifA/cifB expression in males. These findings are consistent with CifB acting as a toxin and CifA as an antitoxin, with CifA attenuating CifB toxicity in both the male germline and in developing embryos. These findings provide important insights into the interactions between cif genes and their mechanism of activity and provide a foundation for the building of a cif gene-based drive system in Ae. aegypti.

CRISPR-based gene drives generate super-Mendelian inheritance in the disease vector Culex quinquefasciatus.

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 a CRISPR-based homing gene drive for Culex quinquefasciatus, and 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-based gene drives generate super-Mendelian inheritance in the disease vector Culex quinquefasciatus.

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.

Closing the gap to effective gene drive in Aedes aegypti by exploiting germline regulatory elements.

CRISPR/Cas9-based homing gene drives have emerged as a potential new approach to mosquito control. While attempts have been made to develop such systems in Aedes aegypti, none have been able to match the high drive efficiency observed in Anopheles species. Here we generate Ae. aegypti transgenic lines expressing Cas9 using germline-specific regulatory elements and assess their ability to bias inheritance of an sgRNA-expressing element (kmosgRNAs). Four shu-Cas9 and one sds3-Cas9 isolines can significantly bias the inheritance of kmosgRNAs, with sds3G1-Cas9 causing the highest average inheritance of ~86% and ~94% from males and females carrying both elements outcrossed to wild-type, respectively. Our mathematical model demonstrates that sds3G1-Cas9 could enable the spread of the kmosgRNAs element to either reach a higher (by ~15 percentage point) maximum carrier frequency or to achieve similar maximum carrier frequency faster (by 12 generations) when compared to two other established split drive systems.

Silencing RNAs expressed from W-linked PxyMasc "retrocopies" target that gene during female sex determination in Plutella xylostella.

The Lepidoptera are an insect order of cultural, economic, and environmental importance, representing ∼10% of all described living species. Yet, for all but one of these species (silkmoth, Bombyx mori), the molecular genetics of how sexual fate is determined remains unknown. We investigated this in the diamondback moth (Plutella xylostella), a globally important, highly invasive, and economically damaging pest of cruciferous crops. Our previous work uncovered a regulator of male sex determination in P. xylostella-PxyMasc, a homolog of B. mori Masculinizer-which, although initially expressed in embryos of both sexes, is then reduced in female embryos, leading to female-specific splicing of doublesex. Here, through sequencing small RNA libraries generated from early embryos and sexed larval pools, we identified a variety of small silencing RNAs (predominantly Piwi-interacting RNAs [piRNAs]) complementary to PxyMasc, whose temporal expression correlated with the reduction in PxyMasc transcript observed previously in females. Analysis of these small RNAs showed that they are expressed from tandemly arranged, multicopy arrays found exclusively on the W (female-specific) chromosome, which we term "Pxyfem". Analysis of the Pxyfem sequences showed that they are partial complementary DNAs (cDNAs) of PxyMasc messenger RNA (mRNA) transcripts, likely integrated into transposable element graveyards by the noncanonical action of retrotransposons (retrocopies), and that their apparent similarity to B. mori feminizer more probably represents convergent evolution. Our study helps elucidate the sex determination cascade in this globally important pest and highlights the "shortcuts" that retrotransposition events can facilitate in the evolution of complex molecular cascades, including sex determination.

Manipulating Insect Sex Determination Pathways for Genetic Pest Management: Opportunities and Challenges.

Sex determination pathways in insects are generally characterised by an upstream primary signal, which is highly variable across species, and that regulates the splicing of a suite of downstream but highly-conserved genes (transformer, doublesex and fruitless). In turn, these downstream genes then regulate the expression of sex-specific characteristics in males and females. Identification of sex determination pathways has and continues to be, a critical component of insect population suppression technologies. For example, "first-generation" transgenic technologies such as fsRIDL (Female-Specific Release of Insects carrying Dominant Lethals) enabled efficient selective removal of females from a target population as a significant improvement on the sterile insect technique (SIT). Second-generation technologies such as CRISPR/Cas9 homing gene drives and precision-guided SIT (pgSIT) have used gene editing technologies to manipulate sex determination genes in vivo. The development of future, third-generation control technologies, such as Y-linked drives, (female to male) sex-reversal, or X-shredding, will require additional knowledge of aspects of sexual development, including a deeper understanding of the nature of primary signals and dosage compensation. This review shows how knowledge of sex determination in target pest species is fundamental to all phases of the development of control technologies.

Toward a CRISPR-Cas9-Based Gene Drive in the Diamondback Moth Plutella xylostella.

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.

Gene drive: a faster route to plant improvement.

Gene drives for control of vector-borne diseases have been demonstrated in insects but remain challenging in plants. Theoretically, they could be transformative in speeding breeding programs and contributing to food security through providing novel weed control methods. Zhang et al. now report the possibility of implementing gene drive in plants for the first time.

CRISPR/Cas-9 mediated knock-in by homology dependent repair in the West Nile Virus vector Culex quinquefasciatus Say.

Culex quinquefasciatus Say is a mosquito distributed in both tropical and subtropical regions of the world. It is a night-active, opportunistic blood-feeder and vectors many animal and human diseases, including West Nile Virus and avian malaria. Current vector control methods (e.g. physical/chemical) are increasingly ineffective; use of insecticides also imposes hazards to both human and ecosystem health. Advances in genome editing have allowed the development of genetic insect control methods, which are species-specific and, theoretically, highly effective. CRISPR/Cas9 is a bacteria-derived programmable gene editing tool that is functional in a range of species. We describe the first successful germline gene knock-in by homology dependent repair in C. quinquefasciatus. Using CRISPR/Cas9, we integrated an sgRNA expression cassette and marker gene encoding a fluorescent protein fluorophore (Hr5/IE1-DsRed, Cq7SK-sgRNA) into the kynurenine 3-monooxygenase (kmo) gene. We achieved a minimum transformation rate of 2.8%, similar to rates in other mosquito species. Precise knock-in at the intended locus was confirmed. Insertion homozygotes displayed a white eye phenotype in early-mid larvae and a recessive lethal phenotype by pupation. This work provides an efficient method for engineering C. quinquefasciatus, providing a new tool for developing genetic control tools for this vector.

Engineered expression of the invertebrate-specific scorpion toxin AaHIT reduces adult longevity and female fecundity in the diamondback moth Plutella xylostella.

BACKGROUND: Previous genetic pest management (GPM) systems in diamondback moth (DBM) have relied on expressing lethal proteins ('effectors') that are 'cell-autonomous', that is, they do not leave the cell in which they are expressed. To increase the flexibility of future GPM systems in DBM, we aimed to assess the use of a non-cell-autonomous, invertebrate-specific, neurotoxic effector - the scorpion toxin AaHIT. This AaHIT effector was designed to be secreted by expressing cells, potentially leading to effects on distant cells, specifically neuromuscular junctions. RESULTS: Expression of AaHIT caused a 'shaking/quivering' phenotype that could be repressed by provision of an antidote (tetracycline): a phenotype consistent with the AaHIT mode-of-action. This effect was more pronounced when AaHIT expression was driven by the Hr5/ie1 promoter (82.44% of males, 65.14% of females) rather than Op/ie2 (57.35% of males, 48.39% of females). Contrary to expectations, the shaking phenotype and observed fitness costs were limited to adults in which they caused severe reductions in mean longevity (-81%) and median female fecundity (-93%). Quantitative polymerase chain reactions of AaHIT expression patterns and analysis of piggyBac-mediated transgene insertion sites suggest that restriction of the observed effects to the adult stages may be due to the influence of the local genomic environment on the tetO-AaHIT transgene. CONCLUSION: We demonstrated the feasibility of using non-cell-autonomous effectors within a GPM context for the first time in Lepidoptera, one of the most economically damaging orders of insects. These findings provide a framework for extending this system to other pest Lepidoptera and to other secreted effectors. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Genetic pest management and the background genetics of release strains.

Genetic pest management (GPM) methods involve releasing modified versions of a pest species to mate with wild pests in the target area. Proposed for a wide range of applications in public health, agriculture and conservation, most progress has been made with pest insects. Offspring of the released modified insects and wild pests carry the modification-which might be transgenes, artificially introduced Wolbachia or genetic damage from radiation, for example-but they also carry a complete haploid genome from their laboratory-reared parent, as well as one from their wild parent. Unless these F1 hybrids are completely unable to reproduce, further mating will lead to introgression of DNA sequences from the release strain into the wild population. We discuss issues around strain selection and the potential consequences of such introgression. We conclude that such introgression is probably harmless in almost all circumstances, and could, in theory, provide specific additional benefits to the release programme. We outline population monitoring approaches that could be used, going forward, to determine how background genetics may affect GPM. This article is part of the theme issue 'Novel control strategies for mosquito-borne diseases'.

Split drive killer-rescue provides a novel threshold-dependent gene drive.

A wide range of gene drive mechanisms have been proposed that are predicted to increase in frequency within a population even when they are deleterious to individuals carrying them. This also allows associated desirable genetic material ("cargo genes") to increase in frequency. Gene drives have garnered much attention for their potential use against a range of globally important problems including vector borne disease, crop pests and invasive species. Here we propose a novel gene drive mechanism that could be engineered using a combination of toxin-antidote and CRISPR components, each of which are already being developed for other purposes. 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.

Ommochrome pathway genes kynurenine 3-hydroxylase and cardinal participate in eye pigmentation in Plutella xylostella.

BACKGROUND: Eye pigmentation genes have been utilized as visible markers for constructing genetic control prototypes in several insect vectors of human disease. Here, orthologs of two ommochrome pathway genes, kynurenine 3-hydroxylase (kmo) and cardinal, were investigated in Plutella xylostella, a globally distributed, economically important pest of Brassica crops. RESULTS: Both somatic mosaic and germline mutations were efficiently created using the CRISPR/Cas9 system, and null mutant strains of Pxkmo and Pxcardinal were obtained. A frame-shift mutation in Pxkmo caused yellow compound eyes at adult stage while an in-frame mutation lacking two amino acids resulted in a hypomorphic red eye phenotypes. In contrast, Pxcardinal-deficient moths with a frame-shift mutation exhibited yellow eye pigmentation in newly emerged adults which turned to red as the adults aged. Additionally, differences were observed in the coloration of larval ocelli, brains and testes in Pxkmo and Pxcardinal yellow-eye mutant lines. CONCLUSIONS: Our work identifies the important roles of Pxkmo and Pxcardinal in P. xylostella eye pigmentation and provides tools for future genetic manipulation of this important crop pest.

Population-level multiplexing: A promising strategy to manage the evolution of resistance against gene drives targeting a neutral locus.

CRISPR-based gene drives bias inheritance in their favour by inducing double-stranded breaks (DSBs) at wild-type homologous loci and using the drive transgene as a repair template-converting drive heterozygotes into homozygotes. Recent studies have shown that alternate end-joining repair mechanisms produce cut-resistant alleles that rapidly induce drive failure. Multiplexing-simultaneously targeting multiple sites at the wild-type locus-is commonly assumed to overcome this issue since resistance would need to develop at all target sites for the system to fail. This may work for some population suppression drives targeting essential (e.g. viability or fertility) genes if careful design can ensure cut-resistant alleles themselves have low fitness. However, here, models are used to demonstrate that this approach will be ineffective when targeting neutral loci. We then go on to compare the performance of four alternative population-level multiplexing approaches with standard individual-level multiplexing. Two of these approaches have mechanisms preventing them from becoming linked, thus avoiding multiple simultaneous DSBs and giving a large improvement. Releasing multiple unlinked drives gives a modest improvement, while releasing multiple drives that may become linked over time produces a decrease in performance under the conditions tested here. Based on performance and technical feasibility, we then take one approach forward for further investigation, demonstrating its robustness to different performance parameters and its potential for controlling very large target populations.

Genetic Biocontrol for Invasive Species.

Invasive species are increasingly affecting agriculture, food, fisheries, and forestry resources throughout the world. As a result of global trade, invasive species are often introduced into new environments where they become established and cause harm to human health, agriculture, and the environment. Prevention of new introductions is a high priority for addressing the harm caused by invasive species, but unfortunately efforts to prevent new introductions do not address the economic harm that is presently manifested where invasive species have already become established. Genetic biocontrol can be defined as the release of organisms with genetic methods designed to disrupt the reproduction of invasive populations. While these methods offer the potential to control or even eradicate invasive species, there is a need to ensure that genetic biocontrol methods can be deployed in a way that minimizes potential harm to the environment. This review provides an overview of the state of genetic biocontrol, focusing on several approaches that were the subject of presentations at the Genetic Biocontrol for Invasive Species Workshop in Tarragona, Spain, March 31st, 2019, a workshop sponsored by the OECD's Co-operative Research Program on Biological Resource Management for Sustainable Agricultural Systems. The review considers four different approaches to genetic biocontrol for invasive species; sterile-release, YY Males, Trojan Female Technique, and gene drive. The different approaches will be compared with respect to the efficiency each affords as a genetic biocontrol tool, the practical utility and cost/benefits associated with implementation of the approach, and the regulatory considerations that will need to be addressed for each. The opinions expressed and arguments employed in this publication are the sole responsibility of the authors and do not necessarily reflect those of the OECD or of the governments of its Member countries.

Identification and characterization of the vasa gene in the diamondback moth, Plutella xylostella.

Vasa is an ATP-dependent RNA helicase, participating in multiple biological processes. It has been widely used as a germ cell marker and its promoter has become a key component of several genetic pest control systems. Here we present the vasa gene structure and its promoter activity in Plutella xylostella, one of the most destructive pests of cruciferous crops. Full length Pxvasa cDNA sequences were obtained, revealing 14 exons and at least 30 alternatively spliced transcripts. Inferred amino acid sequences showed nine conserved DEAD-box family protein motifs with partial exclusion from some isoforms. Real-time quantitative PCR indicated the up-regulation of Pxvasa in both female and male adults compared with other developmental stages, and the expression levels of Pxvasa were found to be much higher in adult gonads, especially ovaries, than in other tissues. The putative promoter region of Pxvasa was sequenced and several ecdysone-induced transcription factor (TF) binding sites were predicted in silico. To further analyze the promoter region, two upstream regulatory fragments of different lengths were tested as putative promoters in transient cell and embryo expression assays, one of which was subsequently utilized to drive Cas9 expression in vivo. A transgenic line was recovered and the expression patterns of Cas9 and native Pxvasa were profiled in adult tissues and eggs with RT-PCR. This work provides the foundation for further studies on the gene functions of Pxvasa as well as the potential application of its promoter in genetic manipulation of P. xylostella.

Expanding the CRISPR Toolbox in Culicine Mosquitoes: In Vitro Validation of Pol III Promoters.

CRISPR-Cas9-based "gene drive" technologies have been proposed as a novel and effective means of controlling human diseases vectored by mosquitoes. However, more complex designs than those demonstrated to date-and an expanded molecular toolbox with which to build them-will be required to overcome the issues of resistance formation/evolution and drive spatial/temporal limitation. Foreseeing this need, we assessed the sgRNA transcriptional activities of 33 phylogenetically diverse insect Polymerase III promoters using three disease-relevant Culicine mosquito cell lines (Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus). We show that U6 promoters work across species with a range of transcriptional activity levels and find 7SK promoters to be especially promising because of their broad phylogenetic activity. We further show that U6 promoters can be substantially truncated without affecting transcriptional levels. These results will be of great utility to researchers involved in developing the next generation of gene drives.

Engineered action at a distance: Blood-meal-inducible paralysis in Aedes aegypti.

BACKGROUND: Population suppression through mass-release of Aedes aegypti males carrying dominant-lethal transgenes has been demonstrated in the field. Where population dynamics show negative density-dependence, suppression can be enhanced if lethality occurs after the density-dependent (i.e. larval) stage. Existing molecular tools have limited current examples of such Genetic Pest Management (GPM) systems to achieving this through engineering 'cell-autonomous effectors' i.e. where the expressed deleterious protein is restricted to the cells in which it is expressed-usually under the control of the regulatory elements (e.g. promoter regions) used to build the system. This limits the flexibility of these technologies as regulatory regions with useful spatial, temporal or sex-specific expression patterns may only be employed if the cells they direct expression in are simultaneously sensitive to existing effectors, and also precludes the targeting of extracellular regions such as cell-surface receptors. Expanding the toolset to 'non-cell autonomous' effectors would significantly reduce these limitations. METHODOLOGY/PRINCIPAL FINDINGS: We sought to engineer female-specific, late-acting lethality through employing the Ae. aegypti VitellogeninA1 promoter to drive blood-meal-inducible, fat-body specific expression of tTAV. Initial attempts using pro-apoptotic effectors gave no evident phenotype, potentially due to the lower sensitivity of terminally-differentiated fat-body cells to programmed-death signals. Subsequently, we dissociated the temporal and spatial expression of this system by engineering a novel synthetic effector (Scorpion neurotoxin-TetO-gp67.AaHIT) designed to be secreted out of the tissue in which it was expressed (fat-body) and then affect cells elsewhere (neuro-muscular junctions). This resulted in a striking, temporary-paralysis phenotype after blood-feeding. CONCLUSIONS/SIGNIFICANCE: These results are significant in demonstrating for the first time an engineered 'action at a distance' phenotype in a non-model pest insect. The potential to dissociate temporal and spatial expression patterns of useful endogenous regulatory elements will extend to a variety of other pest insects and effectors.

Recent advances in threshold-dependent gene drives for mosquitoes.

Mosquito-borne diseases, such as malaria, dengue and chikungunya, cause morbidity and mortality around the world. Recent advances in gene drives have produced control methods that could theoretically modify all populations of a disease vector, from a single release, making whole species less able to transmit pathogens. This ability has caused both excitement, at the prospect of global eradication of mosquito-borne diseases, and concern around safeguards. Drive mechanisms that require individuals to be released at high frequency before genes will spread can therefore be desirable as they are potentially localised and reversible. These include underdominance-based strategies and use of the reproductive parasite Wolbachia Here, we review recent advances in practical applications and mathematical analyses of these threshold-dependent gene drives with a focus on implementation in Aedes aegypti, highlighting their mechanisms and the role of fitness costs on introduction frequencies. Drawing on the parallels between these systems offers useful insights into practical, controlled application of localised drives, and allows us to assess the requirements needed for gene drive reversal.

Towards the genetic control of invasive species.

Invasive species remain one of the greatest threats to global biodiversity. Their control would be enhanced through the development of more effective and sustainable pest management strategies. Recently, a novel form of genetic pest management (GPM) has been developed in which the mating behaviour of insect pests is exploited to introduce genetically engineered DNA sequences into wild conspecific populations. These 'transgenes' work in one or more ways to reduce the damage caused by a particular pest, for example reducing its density, or its ability to vector disease. Although currently being developed for use against economically important insect pests, these technologies would be highly appropriate for application against invasive species that threaten biodiversity. Importantly, these technologies have begun to advance in scope beyond insects to vertebrates, which include some of the world's worst invasives. Here we review the current state of this rapidly progressing field and, using an established set of eradication criteria, discuss the characteristics which make GPM technologies suitable for application against invasive pests.