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Controlling the principal African malaria vector, the mosquito Anopheles gambiae, is considered essential to curtail malaria transmission. However, existing vector control technologies rely on insecticides, which are becoming increasingly ineffective. Sterile insect technique (SIT) is a powerful suppression approach that has successfully eradicated a number of insect pests, yet the A. gambiae toolkit lacks the requisite technologies for its implementation. SIT relies on iterative mass releases of nonbiting, nondriving, sterile males which seek out and mate with monandrous wild females. Once mated, females are permanently sterilized due to mating-induced refractoriness, which results in population suppression of the subsequent generation. However, sterilization by traditional methods renders males unfit, making the creation of precise genetic sterilization methods imperative. Here, we introduce a vector control technology termed precision-guided sterile insect technique (pgSIT), in A. gambiae for inducible, programmed male sterilization and female elimination for wide-scale use in SIT campaigns. Using a binary CRISPR strategy, we cross separate engineered Cas9 and gRNA strains to disrupt male-fertility and female-essential genes, yielding >99.5% male sterility and >99.9% female lethality in hybrid progeny. We demonstrate that these genetically sterilized males have good longevity, are able to induce sustained population suppression in cage trials, and are predicted to eliminate wild A. gambiae populations using mathematical models, making them ideal candidates for release. This work provides a valuable addition to the malaria genetic biocontrol toolkit, enabling scalable SIT-like confinable, species-specific, and safe suppression in the species.

Mosquito-borne illnesses represent a significant global health peril, resulting in approximately one million fatalities annually. West Nile, dengue, Zika, and malaria are continuously expanding their global reach, driven by factors that escalate mosquito populations and pathogen transmission. Innovative control measures are imperative to combat these catastrophic ailments. Conventional approaches, such as eliminating breeding sites and using insecticides, have been helpful, but they face challenges such as insecticide resistance and environmental harm. Given the mounting severity of mosquito-borne diseases, there is promise in exploring innovative approaches using synthetic biology to bolster mosquitoes' resistance to pathogens, or even eliminate the mosquito vectors, as a means of control. This review outlines current strategies, future goals, and the importance of gene editing for global health defenses against mosquito-borne diseases.

Each year, hundreds of millions of people are infected with arboviruses such as dengue, yellow fever, chikungunya, and Zika, which are all primarily spread by the notorious mosquito Aedes aegypti. Traditional control measures have proven insufficient, necessitating innovations. In response, here we generate a next-generation CRISPR-based precision-guided sterile insect technique (pgSIT) for Ae. aegypti that disrupts genes essential for sex determination and fertility, producing predominantly sterile males that can be deployed at any life stage. Using mathematical models and empirical testing, we demonstrate that released pgSIT males can effectively compete with, suppress, and eliminate caged mosquito populations. This versatile species-specific platform has the potential for field deployment to effectively control wild populations of disease vectors.

The resident human skin microbiome is responsible for the production of most of the human scents that are attractive to mosquitoes. Hence, engineering the human skin microbiome to synthesize less of mosquito attractants or produce repellents could potentially reduce bites and prevent the transmission of deadly mosquito-borne pathogens. In order to further characterize the human skin volatilome, we quantified the major volatiles of 39 strains of skin commensals (Staphylococci and Corynebacterium). Importantly, to validate the behavioral activity of these volatiles, we first assessed landing behavior triggered by human skin volatiles. We demonstrated that landing behavior is gated by the presence of carbon dioxide and L-(+)-lactic acid. This is similar to the combinatorial coding triggering mosquito short range attraction. Repellency behavior to selected skin volatiles and terpenes was tested in the presence of carbon dioxide and L-(+)-lactic acid. In a 2-choice landing behavior context, the skin volatiles 2- and 3-methyl butyric acids reduced mosquito landing by 62.0-81.6% and 87.1-99.6%, respectively. Similarly, the terpene geraniol was capable of reducing mosquito landing behavior by 74.9%. We also tested the potential repellency effects of terpenes in mosquitoes at short-range using a 4-port olfactometer. In these assays, geraniol reduced mosquito attraction (69-78%) to a mixture of key human kairomones carbon dioxide, L-(+)-lactic acid, and ammonia. These findings demonstrate that carbon dioxide and L-(+)-lactic acid change the valence of other skin volatiles towards mosquito landing behavior. Moreover, this study offers candidate odorants to be targeted in a novel strategy to reduce attractants or produce repellents by the human skin microbiota that may curtail mosquito bites, and subsequent mosquito-borne disease.

CRISPR diagnostics have recently emerged as powerful diagnostic tools for the rapid detection of infections. The ultimate goal is to develop these diagnostics for the point of care, where patients quickly receive and easily interpret results. Although they are in their infancy, the COVID-19 pandemic has accelerated innovation of CRISPR diagnostics and led to an explosion of improvements to these systems. Challenges that have impeded the implementation at the point of care have been addressed, and CRISPR diagnostics have been dramatically simplified. Here we outline recent developments and advancements in CRISPR diagnostics that have pushed these technologies to the point of care.

Escalating vector disease burdens pose significant global health risks, as such innovative tools for targeting mosquitoes are critical. CRISPR-Cas technologies have played a crucial role in developing powerful tools for genome manipulation in various eukaryotic organisms. Although considerable efforts have focused on utilizing class II type II CRISPR-Cas9 systems for DNA targeting, these modalities are unable to target RNA molecules, limiting their utility against RNA viruses. Recently, the Cas13 family has emerged as an efficient tool for RNA targeting; however, the application of this technique in mosquitoes, particularly Aedes aegypti, has yet to be fully realized. In this study, we engineered an antiviral strategy termed REAPER (vRNA Expression Activates Poisonous Effector Ribonuclease) that leverages the programmable RNA-targeting capabilities of CRISPR-Cas13 and its potent collateral activity. REAPER remains concealed within the mosquito until an infectious blood meal is uptaken. Upon target viral RNA infection, REAPER activates, triggering programmed destruction of its target arbovirus such as chikungunya. Consequently, Cas13-mediated RNA targeting significantly reduces viral replication and viral prevalence of infection, and its promiscuous collateral activity can even kill infected mosquitoes within a few days. This innovative REAPER technology adds to an arsenal of effective molecular genetic tools to combat mosquito virus transmission.

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.

Since the characterization of the CRISPR-Cas9 system in prokaryotes, it has become the prime choice in gene editing because of its exceptional flexibility, ease of use, high efficiency, and superior specificity. As a result, CRISPR-Cas9-mediated gene-editing technologies have enabled researchers not only to engineer transgenic animal strains with site-directed insertions more efficiently but also to generate desired mutants for previously intractable species. One such species is the invasive yellow fever mosquito, Aedes aegypti, which is notorious for its ability to transmit many blood-borne human pathogens. Methods for developing new transgenic strains of the yellow fever mosquito may aid in the effort to control its populations and provide significant benefits for the public. Here, we provide an overview of injection and noninjection methods for generating transgenic mosquitoes and also highlight important experimental design features.

BackgroundDengue virus outbreaks are increasing in number and severity worldwide. Viral transmission is assumed to require a minimum time period of viral replication within the mosquito midgut. It is unknown if alternative transmission periods not requiring replication are possible.MethodsWe used a mouse model of dengue virus transmission to investigate the potential of mechanical transmission of dengue virus. We investigated minimal viral titres necessary for development of symptoms in bitten mice and used resulting parameters to inform a new model of dengue virus transmission within a susceptible population.FindingsNaïve mice bitten by mosquitoes immediately after they took partial blood meals from dengue infected mice showed symptoms of dengue virus, followed by mortality. Incorporation of mechanical transmission into mathematical models of dengue virus transmission suggest that this supplemental transmission route could result in larger outbreaks which peak sooner.InterpretationThe potential of dengue transmission routes independent of midgut viral replication has implications for vector control strategies that target mosquito lifespan and suggest the possibility of similar mechanical transmission routes in other disease-carrying mosquitoes.FundingThis study was funded by grants from the National Health Research Institutes, Taiwan (04D2-MMMOST02), the Human Frontier Science Program (RGP0033/2021), the National Institutes of Health (1R01AI143698-01A1, R01AI151004 and DP2AI152071) and the Ministry of Science and Technology, Taiwan (MOST104-2321-B-400-016).

Malaria is among the world's deadliest diseases, predominantly affecting Sub-Saharan Africa and killing over half a million people annually. Controlling the principal vector, the mosquito Anopheles gambiae, as well as other anophelines, is among the most effective methods to control disease spread. Here, we develop a genetic population suppression system termed Ifegenia (inherited female elimination by genetically encoded nucleases to interrupt alleles) in this deadly vector. In this bicomponent CRISPR-based approach, we disrupt a female-essential gene, femaleless (fle), demonstrating complete genetic sexing via heritable daughter gynecide. Moreover, we demonstrate that Ifegenia males remain reproductively viable and can load both fle mutations and CRISPR machinery to induce fle mutations in subsequent generations, resulting in sustained population suppression. Through modeling, we demonstrate that iterative releases of nonbiting Ifegenia males can act as an effective, confinable, controllable, and safe population suppression and elimination system.

Heat-shock-inducible expression of genes through the use of heat-inducible promoters is commonly used in research despite leaky expression of downstream genes of interest without targeted induction (i.e. heat shock). The development of non-leaky inducible expression systems is of broad interest for both basic and applied studies, to precisely control gene expression. Here we characterize the use of Polycomb response elements and the inducible Heat-shock protein 70Bb promoter, previously described as a non-leaky inducible system, to regulate Cas9 endonuclease levels and function in Drosophila melanogaster after varying both heat-shock durations and rearing temperatures. We show that Polycomb response elements can significantly reduce expression of Cas9 under Heat-shock protein 70Bb promoter control using a range of conditions, corroborating previously published results. We further demonstrate that this low transcript level of heat-induced Cas9 is sufficient to induce mutant mosaic phenotypes. Incomplete suppression of an inducible Cas9 system by Polycomb response elements with no heat-shock suggests that further regulatory elements are required to precisely control Cas9 expression and abundance.

Aedes aegypti Linnaeus and Aedes albopictus Skuse are vectors of dengue virus and responsible for multiple autochthonous dengue outbreaks in Big Island, Hawai'i. Control of Ae. aegypti and Ae. albopictus has been achieved in In2Care trap trials, which motivated us to investigate this potential control approach in the Big Island. Our In2Care trial was performed in the coastal settlement of Miloli'i in the southwest of Big Island where both Ae. aegypti and Ae. albopictus are found. This trial starting in the second week of July and ending in the last week of October 2019 fell within the traditional wet season in Miloli'i. No significant reduction in egg or adult counts in our treatment areas following 12 wk of two In2Care trap placements per participating household were observed. In fact, an increase in numbers of adults during the trial reached levels that required the local mosquito abatement program to stop the In2Care trap trial and institute a thorough source reduction and treatment campaign. The source reduction campaign revealed a large variety and quantity of water sources competed with the oviposition cups we had placed, which likely lowered the chances of our oviposition cups being visited by pyriproxyfen-contaminated Aedes adults exiting the In2Care traps.

Gene-editing technologies have revolutionized the field of mosquito sensory biology. These technologies have been used to knock in reporter genes in-frame with neuronal genes and tag specific mosquito neurons to detect their activities using binary expression systems. Despite these advances, novel tools still need to be developed to elucidate the transmission of olfactory signals from the periphery to the brain. Here, we propose the development of a set of tools, including novel driver lines as well as sensors of neuromodulatory activities, which can advance our knowledge of how sensory input triggers behavioral outputs. This information can change our understanding of mosquito neurobiology and lead to the development of strategies for mosquito behavioral manipulation to reduce bites and disease transmission.

We report the first complete mitogenome (Mt) sequence of Aedes japonicus japonicus (Diptera: Culicidae). The sequence was extracted from one adult from the Big Island of Hawai'i Island. The length of the Ae. japonicus japonicus Mt was 16,528bp with 78.1% AT content. Its sequence is most similar to the Mt sequence of Aedes koreicus with 90.81% sequence identity. This is the first full Mt sequence available for this species and provides important genetic resource for studying population genetics and dynamics of this important invasive mosquito species.

Only female mosquitoes consume blood giving them the opportunity to transmit deadly human pathogens. Therefore, it is critical to remove females before conducting releases for genetic biocontrol interventions. Here we describe a robust sex-sorting approach termed SEPARATOR (Sexing Element Produced by Alternative RNA-splicing of A Transgenic Observable Reporter) that exploits sex-specific alternative splicing of an innocuous reporter to ensure exclusive dominant male-specific expression. Using SEPARATOR, we demonstrate reliable sex selection from early larval and pupal stages in Aedes aegypti, and use a Complex Object Parametric Analyzer and Sorter (COPAS) to demonstrate scalable high-throughput sex-selection of first instar larvae. Additionally, we use this approach to sequence the transcriptomes of early larval males and females and find several genes that are sex-specifically expressed. SEPARATOR can simplify mass production of males for release programs and is designed to be cross-species portable and should be instrumental for genetic biocontrol interventions.

Chemical control of disease vectoring mosquitoes Aedes albopictus and Aedes aegypti is costly, unsustainable, and increasingly ineffective due to the spread of insecticide resistance. The Sterile Insect Technique is a valuable alternative but is limited by slow, error-prone, and wasteful sex-separation methods. Here, we present four Genetic Sexing Strains (two for each Aedes species) based on fluorescence markers linked to the m and M sex loci, allowing for the isolation of transgenic males. Furthermore, we demonstrate how combining these sexing strains enables the production of non-transgenic males. In a mass-rearing facility, 100,000 first instar male larvae could be sorted in under 1.5 h with an estimated 0.01-0.1% female contamination on a single machine. Cost-efficiency analyses revealed that using these strains could result in important savings while setting up and running a mass-rearing facility. Altogether, these Genetic Sexing Strains should enable a major upscaling in control programmes against these important vectors.

We address a controversy over use of the term “gene drive” to include both natural and synthetic genetic elements that promote their own transmission within a population, arguing that this broad definition is both practical and has advantages for risk analysis.

As a major insect vector of multiple arboviruses, Aedes aegypti poses a significant global health and economic burden. A number of genetic engineering tools have been exploited to understand its biology with the goal of reducing its impact. For example, current tools have focused on knocking-down RNA transcripts, inducing loss-of-function mutations, or expressing exogenous DNA. However, methods for transactivating endogenous genes have not been developed. To fill this void, here we developed a CRISPR activation (CRISPRa) system in Ae. aegypti to transactivate target gene expression. Gene expression is activated through pairing a catalytically-inactive ('dead') Cas9 (dCas9) with a highly-active tripartite activator, VP64-p65-Rta (VPR) and synthetic guide RNA (sgRNA) complementary to a user defined target-gene promoter region. As a proof of concept, we demonstrate that engineered Ae. aegypti mosquitoes harboring a binary CRISPRa system can be used to effectively overexpress two developmental genes, even-skipped (eve) and hedgehog (hh), resulting in observable morphological phenotypes. We also used this system to overexpress the positive transcriptional regulator of the Toll immune pathway known as AaRel1, which resulted in a significant suppression of dengue virus serotype 2 (DENV2) titers in the mosquito. This system provides a versatile tool for research pathways not previously possible in Ae. aegypti, such as programmed overexpression of endogenous genes, and may aid in gene characterization studies and the development of innovative vector control tools.

Insect epithelial cells contain unique cellular extensions such as bristles, hairs, and scales. In contrast to bristle and hair, which are not divergent in their shape, scale morphology shows high diversity. In our attempt to characterize the role of the insect-specific gene, Spindle-F (spn-F), in mosquito development, we revealed a scale-type specific requirement for the mosquito Aedes aegypti spn-F homologue. Using CRISPR-Cas9, we generated Ae-spn-F mutants and found that Ae-spn-F is an essential gene, but we were able to recover a few adult escapers. These escapers could not fly nor move, and died after 3 to 4 days. We found that in Ae-spn-F mutants, only the tip part of the bristle was affected with bulbous with misoriented ribs. We also show that in Ae-spn-F mutants, only in falcate scales, which are curved with a sharp or narrowly rounded apex, and not in other scale types, the tip region is strongly affected. Our analysis also revealed that in contrast to Drosophila spn-F, which show strong defects in both the actin and microtubule (MT) network in the bristle, the Ae-spn-F gene is required only for MT organization in scales and bristles. In summary, our results reveal that Ae-spn-F is required for shaping tapered epithelial cellular extension structures, namely, the bristle and falcate scales by affecting MT organization.

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.

Scientists developing gene drive mosquitoes for vector control must understand how residents of affected areas regard both the problem of mosquito-borne disease and the potential solutions offered by gene drive. This study represents an experiment in public engagement at an early stage of technology development, intended to inform lab scientists about public attitudes toward their research and inspire consideration and conversation about the social ramifications of creating mosquitoes with gene drive. Online focus groups with California residents explored views on mosquito-borne disease risk, current mosquito control methods, and the proposed development and use of different classes of gene drives to control Ae. aegypti. Rather than a dogmatic rejection of genetic engineering or gene drive, many participants expressed pragmatic concerns with cost, control, the ability to narrowly target specific species, and the challenges of mistrust and institutional cooperation. Work like this can inform the alignment of community priorities and the professional priorities of scientists and vector control specialists.

Releases of sterile males are the gold standard for many insect population control programs, and precise sex sorting to remove females prior to male releases is essential to the success of these operations. To advance traditional methods for scaling the generation of sterile males, we previously described a CRISPR-mediated precision-guided sterile insect technique (pgSIT), in which Cas9 and gRNA strains are genetically crossed to generate sterile males for mass release. While effective at generating F1 sterile males, pgSIT requires a genetic cross between the two parental strains, which requires maintenance and sexing of two strains in a factory. Therefore, to advance pgSIT further by removing this crossing step, here we describe a next-generation temperature-inducible pgSIT (TI-pgSIT) technology and demonstrate its proof-of-concept in Drosophila melanogaster. Importantly, we were able to develop a true breeding strain for TI-pgSIT that eliminates the requirement for sex sorting-a feature that may help further automate production at scale.

The development of an extensive toolkit for potential point-of-care diagnostics that is expeditiously adaptable to new emerging pathogens is of critical public health importance. Recently, a number of novel CRISPR-based diagnostics have been developed to detect SARS-CoV-2. Herein, we outline the development of an alternative CRISPR nucleic acid diagnostic utilizing a Cas13d ribonuclease derived from Ruminococcus flavefaciens XPD3002 (CasRx) to detect SARS-CoV-2, an approach we term SENSR (sensitive enzymatic nucleic acid sequence reporter) that can detect attomolar concentrations of SARS-CoV-2. We demonstrate 100% sensitivity in patient-derived samples by lateral flow and fluorescence readout with a detection limit of 45 copy/μL. This technology expands the available nucleic acid diagnostic toolkit, which can be adapted to combat future pandemics.

Parasitic nematodes cause significant morbidity and mortality globally. Excretory/secretory products (ESPs) such as fatty acid- and retinol- binding proteins (FARs) are hypothesized to suppress host immunity during nematode infection, yet little is known about their interactions with host tissues. Leveraging the insect parasitic nematode, Steinernema carpocapsae, we describe here the first in vivo study demonstrating that FARs modulate animal immunity, causing an increase in susceptibility to bacterial co-infection. Moreover, we show that FARs dampen key components of the fly immune response including the phenoloxidase cascade and antimicrobial peptide (AMP) production. Our data also reveal that FARs deplete lipid signaling precursors in vivo as well as bind to these fatty acids in vitro, suggesting that FARs elicit their immunomodulatory effects by altering the availability of lipid signaling molecules necessary for an efficient immune response. Collectively, these data support a complex role for FARs in immunosuppression in animals and provide detailed mechanistic insight into parasitism in phylum Nematoda.

The mosquito Aedes aegypti is the principal vector for arboviruses including dengue/yellow fever, chikungunya, and Zika virus, infecting hundreds of millions of people annually. Unfortunately, traditional control methodologies are insufficient, so innovative control methods are needed. To complement existing measures, here we develop a molecular genetic control system termed precision-guided sterile insect technique (pgSIT) in Aedes aegypti. PgSIT uses a simple CRISPR-based approach to generate flightless females and sterile males that are deployable at any life stage. Supported by mathematical models, we empirically demonstrate that released pgSIT males can compete, suppress, and even eliminate mosquito populations. This platform technology could be used in the field, and adapted to many vectors, for controlling wild populations to curtail disease in a safe, confinable, and reversible manner.

In response to growing concerns regarding mosquito-borne diseases, scientists are developing novel systems of vector control. Early examples include Oxitec's OX513A genetically-engineered mosquito and MosquitoMate's Wolbachia-infected mosquito, and systems using 'gene-drive' are in development. Systems based on genetic engineering are controversial and institutions around the world are grappling with the question of who should have a say in how such technologies are field-tested and used. Based on media coverage and public records, we created comparative timelines of the efforts of Oxitec and MosquitoMate to navigate federal and local governance and bring their products to market in the United States. We analyze these timelines with particular attention to the role of public input in technology governance. These cases illustrate how governance of technology in the US is diverse, complex, and opaque. Further, the public response to proposed field trials of the Oxitec product highlights inconsistencies between public expectations for governance and actual practice. As gene-drive mosquito control products develop, both federal and local agencies will find their legitimacy tested without a better procedure for transparently integrating public input.

Mosquito-borne diseases, such as dengue and malaria, pose significant global health burdens. Unfortunately, current control methods based on insecticides and environmental maintenance have fallen short of eliminating the disease burden. Scalable, deployable, genetic-based solutions are sought to reduce the transmission risk of these diseases. Pathogen-blocking Wolbachia bacteria, or genome engineering-based mosquito control strategies including gene drives have been developed to address these problems, both requiring the release of modified mosquitoes into the environment. Here, we review the latest developments, notable similarities, and critical distinctions between these promising technologies and discuss their future applications for mosquito-borne disease control.

Engineered reproductive species barriers are useful for impeding gene flow and driving desirable genes into wild populations in a reversible threshold-dependent manner. However, methods to generate synthetic barriers are lacking in advanced eukaryotes. Here, to overcome this challenge, we engineer SPECIES (Synthetic Postzygotic barriers Exploiting CRISPR-based Incompatibilities for Engineering Species), an engineered genetic incompatibility approach, to generate postzygotic reproductive barriers. Using this approach, we create multiple reproductively isolated SPECIES and demonstrate their reproductive isolation and threshold-dependent gene drive capabilities in D. melanogaster. Given the near-universal functionality of CRISPR tools, this approach should be portable to many species, including insect disease vectors in which confinable gene drives could be of great practical utility.

Aedes aegypti spread devastating viruses such as dengue, which causes disease among 100 to 400 million people annually. A potential approach to control mosquito disease vectors is the sterile insect technique (SIT). The strategy involves repeated release of large numbers of sterile males, which reduces insect populations because the sterile males mate and thereby suppress the fertility of females that would otherwise mate with fertile males. While SIT has been successful in suppressing certain agricultural pests, it has been less effective in depressing populations of Ae. aegypti This limitation is in part because of the fitness effects resulting from mutagenizing the mosquitoes nonspecifically. Here, we introduced and characterized the impact on female fertility of an Ae. aegypti mutation that disrupts a gene that is specifically expressed in testes. We used CRISPR/Cas9 to generate a null mutation in the Ae. aegypti β2-tubulin (B2t) gene, which eliminates male fertility. When we allowed wild-type females to first mate with B2t mutant males, most of the females did not produce progeny even after being subsequently exposed to wild-type males. We also introduced B2t mutant and wild-type males simultaneously with wild-type females and found that a larger number of B2t mutant males relative to the wild-type males was effective in significantly suppressing female fertility. These results raise the possibility of employing B2t sterile males to improve the efficacy of SIT in suppressing populations of Ae. aegypti through repeated releases and thereby reduce the transmission of viruses by these invasive mosquitoes.

Insects naturally harbor a broad range of selfish agents that can manipulate their reproduction and development, often leading to host sex ratio distortion. Such effects directly benefit the spread of the selfish agents. These agents include two broad groups: bacterial symbionts and selfish chromosomes. Recent studies have made steady progress in uncovering the cellular targets of these agents and their effector genes. Here we highlight what is known about the targeted developmental processes, developmental timing, and effector genes expressed by several selfish agents. It is now becoming apparent that: (1) the genetic toolkits used by these agents to induce a given reproductive manipulation are simple, (2) these agents target sex-specific cellular processes very early in development, and (3) in some cases, similar processes are targeted. Knowledge of the molecular underpinnings of these systems will help to solve long-standing puzzles and provide new tools for controlling insect pests.

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. Finally, we conduct mathematical modeling comparing HomeR to contemporary gene drive architectures for population modification over wide ranges of fitness costs, transmission rates, and release regimens. HomeR could potentially be adapted to other species, as a means for safe, confinable, modification of wild populations.

CRISPR-based gene-drive systems, which copy themselves via gene conversion mediated by the homology-directed repair (HDR) pathway, have the potential to revolutionize vector control. However, mutant alleles generated by the competing non-homologous end-joining (NHEJ) pathway, resistant to Cas9 cleavage, can interrupt the spread of gene-drive elements. We hypothesized that drives targeting genes essential for viability or reproduction also carrying recoded sequences that restore endogenous gene functionality should benefit from dominantly-acting maternal clearance of NHEJ alleles combined with recessive Mendelian culling processes. Here, we test split gene-drive (sGD) systems in Drosophila melanogaster that are inserted into essential genes required for viability (rab5, rab11, prosalpha2) or fertility (spo11). In single generation crosses, sGDs copy with variable efficiencies and display sex-biased transmission. In multigenerational cage trials, sGDs follow distinct drive trajectories reflecting their differential tendencies to induce target chromosome damage and/or lethal/sterile mosaic Cas9-dependent phenotypes, leading to inherently confinable drive outcomes.

Although mosquitoes are major transmission vectors for pathogenic arboviruses, viral infection has little impact on mosquito health. This immunity is caused in part by mosquito RNA interference (RNAi) pathways that generate antiviral small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). RNAi also maintains genome integrity by potently repressing mosquito transposon activity in the germline and soma. However, viral and transposon small RNA regulatory pathways have not been systematically examined together in mosquitoes. Therefore, we developed an integrated mosquito small RNA genomics (MSRG) resource that analyzes the transposon and virus small RNA profiles in mosquito cell cultures and somatic and gonadal tissues across four medically important mosquito species. Our resource captures both somatic and gonadal small RNA expression profiles within mosquito cell cultures, and we report the evolutionary dynamics of a novel Mosquito-Conserved piRNA Cluster Locus (MCpiRCL) made up of satellite DNA repeats. In the larger culicine mosquito genomes we detected highly regular periodicity in piRNA biogenesis patterns coinciding with the expansion of Piwi pathway genes. Finally, our resource enables detection of cross talk between piRNA and siRNA populations in mosquito cells during a response to virus infection. The MSRG resource will aid efforts to dissect and combat the capacity of mosquitoes to tolerate and spread arboviruses.

CasRx, a member of the RNA-targeting Cas13 family, is a promising new addition of the CRISPR/Cas technologies in efficient gene transcript reduction with an attractive off-target profile at both cellular and organismal levels. It is recently reported that the CRISPR/CasRx system can be used to achieve ubiquitous and tissue-specific gene transcript reduction in Drosophila melanogaster. This paper details the methods from the recent work, consisting of three parts: 1) ubiquitous in vivo endogenous RNA targeting using a two-component CasRx system; 2) ubiquitous in vivo exogenous RNA targeting using a three-component CasRx system; and 3) tissue-specific in vivo RNA targeting using a three-component CasRx system. The effects of RNA targeting observed include targeted gene specific phenotypic changes, targeted RNA transcript reduction, and occasional lethality phenotypes associated with high expression of CasRx protein and collateral activity. Overall, these results showed that the CasRx system is capable of target RNA transcript reduction at the organismal level in a programmable and efficient manner, demonstrating that in vivo transcriptome targeting, and engineering is feasible and lays the foundation for future in vivo CRISPR-based RNA targeting technologies.

The areas where dengue virus (DENV) is endemic have expanded rapidly, driven in part by the global spread of Aedes species, which act as disease vectors. DENV replicates in the mosquito midgut and is disseminated to the mosquito's salivary glands for amplification. Thus, blocking virus infection or replication in the tissues of the mosquito may be a viable strategy for reducing the incidence of DENV transmission to humans. Here we used the mariner Mos1 transposase to create an Aedes aegypti line that expresses virus-specific miRNA hairpins capable of blocking DENV replication. These microRNA are driven by the blood-meal-inducible carboxypeptidase A promoter or by the polyubiquitin promoter. The transgenic mosquitoes exhibited significantly lower infection rates and viral titers for most DENV serotypes 7 days after receiving an infectious blood meal. The treatment was also effective at day 14 post infection after a second blood meal had been administered. In viral transmission assay, we found there was significantly reduced transmission in these lines. These transgenic mosquitoes were effective in silencing most of the DENV genome; such an approach may be employed to control a dengue fever epidemic.

CRISPR-based genetic engineering tools aimed to bias sex ratios, or drive effector genes into animal populations, often integrate the transgenes into autosomal chromosomes. However, in species with heterogametic sex chromsomes (e.g. XY, ZW), sex linkage of endonucleases could be beneficial to drive the expression in a sex-specific manner to produce genetic sexing systems, sex ratio distorters, or even sex-specific gene drives, for example. To explore this possibility, here we develop a transgenic line of Drosophila melanogaster expressing Cas9 from the Y chromosome. We functionally characterize the utility of this strain for both sex selection and gene drive finding it to be quite effective. To explore its utility for population control, we built mathematical models illustrating its dynamics as compared to other state-of-the-art systems designed for both population modification and suppression. Taken together, our results contribute to the development of current CRISPR genetic control tools and demonstrate the utility of using sex-linked Cas9 strains for genetic control of animals.

Aedes aegypti Act4 is a paralog of the Drosophila melanogaster indirect flight muscle actin gene Act88F. Act88F has been shown to be haploinsufficient for flight in both males and females (amorphic mutants are dominant). Whereas Act88F is expressed in indirect flight muscles of both males and females, expression of Act4 is substantially female-specific. We therefore used CRISPR/Cas9 and homology directed repair to examine the phenotype of Act4 mutants in two Culicine mosquitoes, Aedes aegypti and Culex quinquefasciatus. A screen for dominant female-flightless mutants in Cx. quinquefasciatus identified one such mutant associated with a six base pair deletion in the CxAct4 coding region. A similar screen in Ae. aegypti identified no dominant mutants. Disruption of the AeAct4 gene by homology-dependent insertion of a fluorescent protein marker cassette gave a recessive female-flightless phenotype in Ae. aegypti. Reproducing the six-base deletion from Cx. quinquefasciatus in Ae. aegypti using oligo-directed mutagenesis generated dominant female-flightless mutants and identified additional dominant female-flightless mutants with other in-frame insertions or deletions. Our data indicate that loss of function mutations in the AeAct4 gene are recessive but that short in-frame deletions produce dominant-negative versions of the AeAct4 protein that interfere with flight muscle function. This makes Act4 an interesting candidate for genetic control methods, particularly population-suppression gene drives targeting female viability/fertility.

BackgroundThe Asian tiger mosquito Aedes albopictus is globally expanding and has become the main vector for human arboviruses in Europe. With limited antiviral drugs and vaccines available, vector control is the primary approach to prevent mosquito-borne diseases. A reliable and accurate DNA sequence of the Ae. albopictus genome is essential to develop new approaches that involve genetic manipulation of mosquitoes.ResultsWe use long-read sequencing methods and modern scaffolding techniques (PacBio, 10X, and Hi-C) to produce AalbF2, a dramatically improved assembly of the Ae. albopictus genome. AalbF2 reveals widespread viral insertions, novel microRNAs and piRNA clusters, the sex-determining locus, and new immunity genes, and enables genome-wide studies of geographically diverse Ae. albopictus populations and analyses of the developmental and stage-dependent network of expression data. Additionally, we build the first physical map for this species with 75% of the assembled genome anchored to the chromosomes.ConclusionThe AalbF2 genome assembly represents the most up-to-date collective knowledge of the Ae. albopictus genome. These resources represent a foundation to improve understanding of the adaptation potential and the epidemiological relevance of this species and foster the development of innovative control measures.

BackgroundWhile there is wide consensus that the public should be consulted about emerging technology early in development, it is difficult to elicit public opinion about innovations unfamiliar to lay audiences. We sought public input on a program of research on genetic engineering to control mosquito vectors of disease that is led by scientists at the University of California and funded by the U.S. Defense Advanced Research Projects Agency (DARPA). In preparation for this effort, we developed a series of narrated slideshows to prompt responses to the development of gene drive mosquito control strategies among lay people. We describe the development and content of these slideshows and evaluate their ability to elicit discussions among focus group participants.MethodsIn developing these materials, we used an iterative process involving input from experts in molecular genetics and vector control. Topics were chosen for their relevance to the goals of the scientists leading the program of research. Significant time was devoted to crafting explanations that would be accessible to uninitiated members of the public but still represent the science accurately. Through qualitative analysis of focus group discussions prompted by the slideshows, we evaluated the success of these slideshows in imparting clear technical information sufficient to inform lay discussion.ResultsThe collaboration resulted in a series of four narrated slideshows that were used to anchor discussions in online focus groups. Many participants described the slideshows as interesting and informative, while also raising concerns and possible risks that were not directly addressed in the material presented. Open-ended comments from participants suggest that the slideshows inspired critical questions, reflection, and conversation about genetically engineered and gene drive mosquitoes. After the final and most technically complex slideshow, however, some respondents made comments suggestive of overwhelm or confusion.ConclusionOur narrated slideshows prompted engaged conversations about genetically engineered mosquitoes among members of the public who were generally naïve to this technology. Narrated slideshows may serve as viable and useful tools for future public engagement on other controversial emerging medical and public health technologies.

BackgroundThe discovery of CRISPR-based gene editing and its application to homing-based gene drive systems has been greeted with excitement, for its potential to control mosquito-borne diseases on a wide scale, and concern, for the invasiveness and potential irreversibility of a release. Gene drive systems that display threshold-dependent behavior could potentially be used during the trial phase of this technology, or when localized control is otherwise desired, as simple models predict them to spread into partially isolated populations in a confineable manner, and to be reversible through releases of wild-type organisms. Here, we model hypothetical releases of two recently engineered threshold-dependent gene drive systems-reciprocal chromosomal translocations and a form of toxin-antidote-based underdominance known as UDMEL-to explore their ability to be confined and remediated.ResultsWe simulate releases of Aedes aegypti, the mosquito vector of dengue, Zika, and other arboviruses, in Yorkeys Knob, a suburb of Cairns, Australia, where previous biological control interventions have been undertaken on this species. We monitor spread to the neighboring suburb of Trinity Park to assess confinement. Results suggest that translocations could be introduced on a suburban scale, and remediated through releases of non-disease-transmitting male mosquitoes with release sizes on the scale of what has been previously implemented. UDMEL requires fewer releases to introduce, but more releases to remediate, including of females capable of disease transmission. Both systems are expected to be confineable to the release site; however, spillover of translocations into neighboring populations is less likely.ConclusionsOur analysis supports the use of translocations as a threshold-dependent drive system capable of spreading disease-refractory genes into Ae. aegypti populations in a confineable and reversible manner. It also highlights increased release requirements when incorporating life history and population structure into models. As the technology nears implementation, further ecological work will be essential to enhance model predictions in preparation for field trials.

We must ensure that trials are scientifically, politically, and socially robust, publicly accountable, and widely transparent

Information about genetic engineering (GE) for vector control in the United States is disseminated primarily in English, though non-English speakers are equally, and in some geographic regions even more affected by such technologies. Non-English-speaking publics should have equal access to such information, which is especially critical when the technology in question may impact whole communities. We convened an interdisciplinary workgroup to translate previously developed narrated slideshows on gene drive mosquitoes from English into Spanish, reviewing each iteration for scientific accuracy and accessibility to laypeople. Using the finalised stimuli, we conducted five online, chat-based focus groups with Spanish-speaking adults from California. Overall, participants expressed interest in the topic and were able to summarise the information presented in their own words. Importantly, participants asked for clarification and expressed scepticism about the information presented, indicating critical engagement with the material. Through collaboration with Spanish-speaking scientists engaged in the development of GE methods of vector control, we translated highly technical scientific information into Spanish that successfully engaged Spanish-speaking participants in conversations about this topic. In this manuscript, we document the feasibility of consulting Spanish-speaking publics about a complex emerging technology by drawing on the linguistic diversity of the scientific teams developing the technology.

CRISPR-Cas9-based gene drive systems possess the inherent capacity to spread progressively throughout target populations. 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). e-CHACRs can be inserted at various genomic locations and carry two or more gRNAs, the first copying the e-CHACR and the second mutating and inactivating the cas9 transgene. Alternatively, ERACRs are inserted at the same genomic location as a gene drive, carrying two gRNAs that cut on either side of the gene drive to excise it. e-CHACRs efficiently inactivate Cas9 and can drive to completion in cage experiments. Similarly, ERACRs, particularly those carrying a recoded cDNA-restoring endogenous gene activity, can drive reliably to fully replace a gene drive. We compare the strengths of these two systems.

Animals face the dual threat of virus infections hijacking cellular function and transposons proliferating in germline genomes. For insects, the deeply conserved RNA interference (RNAi) pathways and other chromatin regulators provide an important line of defense against both viruses and transposons. For example, this innate immune system displays adaptiveness to new invasions by generating cognate small RNAs for targeting gene silencing measures against the viral and genomic intruders. However, within the Dipteran clade of insects, Drosophilid fruit flies and Culicids mosquitoes have evolved several unique mechanistic aspects of their RNAi defenses to combat invading transposons and viruses, with the Piwi-piRNA arm of the RNAi pathways showing the greatest degree of novel evolution. Whereas central features of Piwi-piRNA pathways are conserved between Drosophilids and Culicids, multiple lineage-specific innovations have arisen that may reflect distinct genome composition differences and specific ecological and physiological features dividing these two branches of Dipterans. This perspective review focuses on the most recent findings illuminating the Piwi/piRNA pathway distinctions between fruit flies and mosquitoes, and raises open questions that need to be addressed in order to ameliorate human diseases caused by pathogenic viruses that mosquitoes transmit as vectors.

CRISPR-Cas genome editing technologies have revolutionized the fields of functional genetics and genome engineering, but with the recent discovery and optimization of RNA-targeting Cas ribonucleases, we may soon see a similar revolution in the study of RNA function and transcriptome engineering. However, to date, successful proof of principle for Cas ribonuclease RNA targeting in eukaryotic systems has been limited. Only recently has successful modification of RNA expression by a Cas ribonuclease been demonstrated in animal embryos. This previous work, however, did not evaluate endogenous expression of Cas ribonucleases and only focused on function in early developmental stages. A more comprehensive evaluation of this technology is needed to assess its potential impact. Here we report on our efforts to develop a programmable platform for RNA targeting using a Cas ribonuclease, CasRx, in the model organism Drosophila melanogaster. By genetically encoding CasRx in flies, we demonstrate moderate transcript targeting of known phenotypic genes in addition to unexpected toxicity and lethality. We also report on the off-target effects following on-target transcript cleavage by CasRx. Taken together, our results present the current state and limitations of a genetically encoded programmable RNA-targeting Cas system in Drosophila melanogaster, paving the way for future optimization of the system.

Here, we describe a drug-inducible genetic system for insect sex-separation that demonstrates proof-of-principle for positive sex selection in D. melanogaster. The system exploits the toxicity of commonly used broad-spectrum antibiotics geneticin and puromycin to kill the non-rescued sex. Sex-specific rescue is achieved by inserting sex-specific introns into the coding sequences of antibiotic-resistance genes. When raised on geneticin-supplemented food, the sex-sorter line establishes 100% positive selection for female progeny, while the food supplemented with puromycin positively selects 100% male progeny. Since the described system exploits conserved sex-specific splicing mechanisms and reagents, it has the potential to be adaptable to other insect species of medical and agricultural importance.