RNA interference is essential to modulating the pathogenesis of mosquito-borne viruses in the yellow fever mosquito Aedes aegypti.

While it has long been known that the transmission of mosquito-borne viruses depends on the establishment of persistent and nonlethal infections in the invertebrate host, specific roles for the insects' antiviral immune pathways in modulating the pathogenesis of viral infections is the subject of speculation and debate. Here, we show that a loss-of-function mutation in the Aedes aegypti Dicer-2 (Dcr-2) gene renders the insect acutely susceptible to a disease phenotype upon infection with pathogens in multiple virus families associated with important human diseases. Additional interrogation of the disease phenotype demonstrated that the virus-induced pathology is controlled through a canonical RNA interference (RNAi) pathway, which functions as a resistance mechanism. These results suggest comparatively modest contributions of proposed tolerance mechanisms to the fitness of A. aegypti infected with these pathogens. Similarly, the production of virus-derived piwi-interacting RNAs (vpiRNAs) was not sufficient to prevent the pathology associated with viral infections in Dcr-2 null mutants, also suggesting a less critical, or potentially secondary, role for vpiRNAs in antiviral immunity. These findings have important implications for understanding the ecological and evolutionary interactions occurring between A. aegypti and the pathogens they transmit to human and animal hosts.

Novel synthetic 3'-untranslated regions for controlling transgene expression in transgenic Aedes aegypti mosquitoes.

Transgenic technology for mosquitoes is now more than two decades old, and a wide array of control sequences have been described for regulating gene expression in various life stages or specific tissues. Despite this, comparatively little attention has been paid to the development and validation of other transgene-regulating elements, especially 3'-untranslated regions (3'UTRs). As a consequence, the same regulatory sequences are often used multiple times in a single transgene array, potentially leading to instability of transgenic effector genes. To increase the repertoire of characterized 3'UTRs available for genetics-based mosquito control, we generated fifteen synthetic sequences based on the base composition of the widely used SV40 3'UTR sequence, and tested their ability to contribute to the expression of reporter genes EGFP or luciferase. Transient transfection in mosquito cells identified nine candidate 3'UTRs that conferred moderate to strong gene expression. Two of these were engineered into the mosquito genome through CRISPR/Cas9-mediated site-specific insertion and compared to the original SV40 3'UTR. Both synthetic 3'UTRs were shown to successfully promote transgene expression in all mosquito life stages (larva, pupa and adults), similar to the SV40 3'UTR, albeit with differences in intensity. Thus, the synthetic 3'UTR elements described here are suitable for regulating transgene expression in Ae. aegypti, and provide valuable alternatives in the design of multi-gene cassettes. Additionally, the synthetic-scramble approach we validate here could be used to generate additional functional 3'UTR elements in this or other organisms.

Yellow fever virus capsid protein is a potent suppressor of RNA silencing that binds double-stranded RNA.

Mosquito-borne flaviviruses, including yellow fever virus (YFV), Zika virus (ZIKV), and West Nile virus (WNV), profoundly affect human health. The successful transmission of these viruses to a human host depends on the pathogen's ability to overcome a potentially sterilizing immune response in the vector mosquito. Similar to other invertebrate animals and plants, the mosquito's RNA silencing pathway comprises its primary antiviral defense. Although a diverse range of plant and insect viruses has been found to encode suppressors of RNA silencing, the mechanisms by which flaviviruses antagonize antiviral small RNA pathways in disease vectors are unknown. Here we describe a viral suppressor of RNA silencing (VSR) encoded by the prototype flavivirus, YFV. We show that the YFV capsid (YFC) protein inhibits RNA silencing in the mosquito Aedes aegypti by interfering with Dicer. This VSR activity appears to be broadly conserved in the C proteins of other medically important flaviviruses, including that of ZIKV. These results suggest that a molecular "arms race" between vector and pathogen underlies the continued existence of flaviviruses in nature.

Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis in Aedes aegypti.

Conventional control strategies for mosquito-borne pathogens such as malaria and dengue are now being complemented by the development of transgenic mosquito strains reprogrammed to generate beneficial phenotypes such as conditional sterility or pathogen resistance. The widespread success of site-specific nucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 in model organisms also suggests that reprogrammable gene drive systems based on these nucleases may be capable of spreading such beneficial phenotypes in wild mosquito populations. Using the mosquito Aedes aegypti, we determined that mutations in the FokI domain used in TALENs to generate obligate heterodimeric complexes substantially and significantly reduce gene editing rates. We found that CRISPR/Cas9-based editing in the mosquito Ae. aegypti is also highly variable, with the majority of guide RNAs unable to generate detectable editing. By first evaluating candidate guide RNAs using a transient embryo assay, we were able to rapidly identify highly effective guide RNAs; focusing germ line-based experiments only on this cohort resulted in consistently high editing rates of 24-90%. Microinjection of double-stranded RNAs targeting ku70 or lig4, both essential components of the end-joining response, increased recombination-based repair in early embryos as determined by plasmid-based reporters. RNAi-based suppression of Ku70 concurrent with embryonic microinjection of site-specific nucleases yielded consistent gene insertion frequencies of 2-3%, similar to traditional transposon- or ΦC31-based integration methods but without the requirement for an initial docking step. These studies should greatly accelerate investigations into mosquito biology, streamline development of transgenic strains for field releases, and simplify the evaluation of novel Cas9-based gene drive systems.

The hub protein loquacious connects the microRNA and short interfering RNA pathways in mosquitoes.

Aedes aegypti mosquitoes vector several arboviruses of global health significance, including dengue viruses and chikungunya virus. RNA interference (RNAi) plays an important role in antiviral immunity, gene regulation and protection from transposable elements. Double-stranded RNA binding proteins (dsRBPs) are important for efficient RNAi; in Drosophila functional specialization of the miRNA, endo-siRNA and exo-siRNA pathway is aided by the dsRBPs Loquacious (Loqs-PB, Loqs-PD) and R2D2, respectively. However, this functional specialization has not been investigated in other dipterans. We were unable to detect Loqs-PD in Ae. aegypti; analysis of other dipteran genomes demonstrated that this isoform is not conserved outside of Drosophila. Overexpression experiments and small RNA sequencing following depletion of each dsRBP revealed that R2D2 and Loqs-PA cooperate non-redundantly in siRNA production, and that these proteins exhibit an inhibitory effect on miRNA levels. Conversely, Loqs-PB alone interacted with mosquito dicer-1 and was essential for full miRNA production. Mosquito Loqs interacted with both argonaute 1 and 2 in a manner independent of its interactions with dicer. We conclude that the functional specialization of Loqs-PD in Drosophila is a recently derived trait, and that in other dipterans, including the medically important mosquitoes, Loqs-PA participates in both the miRNA and endo-siRNA based pathways.

Understanding the DNA damage response in order to achieve desired gene editing outcomes in mosquitoes.

Mosquitoes are high-impact disease vectors with the capacity to transmit pathogenic agents that cause diseases such as malaria, yellow fever, chikungunya, and dengue. Continued growth in knowledge of genetic, molecular, and physiological pathways in mosquitoes allows for the development of novel control methods and for the continued optimization of existing ones. The emergence of site-specific nucleases as genomic engineering tools promises to expedite research of crucial biological pathways in these disease vectors. The utilization of these nucleases in a more precise and efficient manner is dependent upon knowledge and manipulation of the DNA repair pathways utilized by the mosquito. While progress has been made in deciphering DNA repair pathways in some model systems, research into the nature of the hierarchy of mosquito DNA repair pathways, as well as in mechanistic differences that may exist, is needed. In this review, we will describe progress in the use of site-specific nucleases in mosquitoes, along with the hierarchy of DNA repair in the context of mosquito chromosomal organization and structure, and how this knowledge may be manipulated to achieve precise chromosomal engineering in mosquitoes.

Targeted genome editing in Aedes aegypti using TALENs.

The Culicine mosquito, Aedes aegypti, is both a major vector of arthropod-borne viruses (arboviruses) and a genetic model organism for arbovirus transmission. TALE nucleases (TALENs), a group of artificial enzymes capable of generating site-specific DNA lesions, consist of a non-specific FokI endonuclease cleavage domain fused to an engineered DNA binding domain specific to a target site. While TALENs have become an important tool for targeted gene disruption in a variety of organisms, application to the mosquito genome is a new approach. We recently described the use of TALENs to perform heritable genetic disruptions in A. aegypti. Here, we provide detailed methods that will allow other research laboratories to capitalize on the potential of this technology for understanding mosquito gene function. We describe target site selection, transient embryo-based assays to rapidly assess TALEN activity, embryonic microinjection and downstream screening steps to identify target site mutations.

Production of virus-derived ping-pong-dependent piRNA-like small RNAs in the mosquito soma.

The natural maintenance cycles of many mosquito-borne pathogens require establishment of persistent non-lethal infections in the invertebrate host. The mechanism by which this occurs is not well understood, but we have previously shown that an antiviral response directed by small interfering RNAs (siRNAs) is important in modulating the pathogenesis of alphavirus infections in the mosquito. However, we report here that infection of mosquitoes with an alphavirus also triggers the production of another class of virus-derived small RNAs that exhibit many similarities to ping-pong-dependent piwi-interacting RNAs (piRNAs). However, unlike ping-pong-dependent piRNAs that have been described previously from repetitive elements or piRNA clusters, our work suggests production in the soma. We also present evidence that suggests virus-derived piRNA-like small RNAs are capable of modulating the pathogenesis of alphavirus infections in dicer-2 null mutant mosquito cell lines defective in viral siRNA production. Overall, our results suggest that a non-canonical piRNA pathway is present in the soma of vector mosquitoes and may be acting redundantly to the siRNA pathway to target alphavirus replication.

Transgene removal using an in cis programmed homing endonuclease via single-strand annealing in the mosquito Aedes aegypti.

While gene drive strategies have been proposed to aid in the control of mosquito-borne diseases, additional genome engineering technologies may be required to establish a defined end-of-product-life timeline. We previously demonstrated that single-strand annealing (SSA) was sufficient to program the scarless elimination of a transgene while restoring a disrupted gene in the disease vector mosquito Aedes aegypti. Here, we extend these findings by establishing that complete transgene removal (four gene cassettes comprising ~8-kb) can be programmed in cis. Reducing the length of the direct repeat from 700-bp to 200-bp reduces, but does not eliminate, SSA activity. In contrast, increasing direct repeat length to 1.5-kb does not increase SSA rates, suggesting diminishing returns above a certain threshold size. Finally, we show that while the homing endonuclease Y2-I-AniI triggered both SSA and NHEJ at significantly higher rates than I-SceI at one genomic locus (P5-EGFP), repair events are heavily skewed towards NHEJ at another locus (kmo), suggesting the nuclease used and the genomic region targeted have a substantial influence on repair outcomes. Taken together, this work establishes the feasibility of engineering temporary transgenes in disease vector mosquitoes, while providing critical details concerning important operational parameters.

Expansions to the MGDrivE suite for simulating the efficacy of novel gene-drive constructs in the control of mosquito-borne diseases.

OBJECTIVE: The MGDrivE (MGDrivE 1 and MGDrivE 2) modeling framework provides a flexible and expansive environment for testing the efficacy of novel gene-drive constructs for the control of mosquito-borne diseases. However, the existing model framework did not previously support several features necessary to simulate some types of intervention strategies. Namely, current MGDrivE versions do not permit modeling of small molecule inducible systems for controlling gene expression in gene drive designs or the inheritance patterns of self-eliminating gene drive mechanisms. RESULTS: Here, we demonstrate a new MGDrivE 2 module that permits the simulation of gene drive strategies incorporating small molecule-inducible systems and self-eliminating gene drive mechanisms. Additionally, we also implemented novel sparsity-aware sampling algorithms for improved computational efficiency in MGDrivE 2 and supplied an analysis and plotting function applicable to the outputs of MGDrivE 1 and MGDrivE 2.

CRISPR-based gene editing of non-homologous end joining factors biases DNA repair pathway choice toward single-strand annealing in Aedes aegypti.

To maintain genome stability, eukaryotic cells orchestrate DNA repair pathways to process DNA double-strand breaks (DSBs) that result from diverse developmental or environmental stimuli. Bias in the selection of DSB repair pathways, either non-homologous end joining (NHEJ) or homology-directed repair (HDR), is also critical for efficient gene editing and for homing-based gene drive approaches developed for the control of disease-transmitting vector mosquitoes. However, little is understood about DNA repair homeostasis in the mosquito genome. Here, we utilized CRISPR/Cas9 to generate indel mutant strains for core NHEJ factors ku80, DNA ligase IV (lig4), and DNA-PKcs in the mosquito Aedes aegypti and evaluated the corresponding effects on DNA repair. In a plasmid-based assay, disruption of ku80 or lig4, but not DNA-PKcs, reduced both NHEJ and SSA. However, a transgenic reporter strain-based test revealed that those mutations significantly biased DNA repair events toward SSA. Interestingly, ku80 mutation also significantly increased the end joining rate by a yet-characterized mechanism in males. Our study provides evidence that the core NHEJ factors have an antagonistic effect on SSA-based DSB repair of the Ae. aegypti genome. Down-modulating the NHEJ pathway can enhance the efficiency of nuclease-based genetic control approaches, as most of those operate by homology-based repair processes along with extensive DNA end resection that is antagonized by NHEJ.

Repeat mediated excision of gene drive elements for restoring wild-type populations.

We demonstrate here that single strand annealing (SSA) repair can be co-opted for the precise autocatalytic excision of a drive element. Although SSA is not the predominant form of DNA repair in eukaryotic organisms, we increased the likelihood of its use by engineering direct repeats at sites flanking the drive allele, and then introducing a double-strand DNA break (DSB) at a second endonuclease target site encoded within the drive allele. We have termed this technology Repeat Mediated Excision of a Drive Element (ReMEDE). Incorporation of ReMEDE into the previously described mutagenic chain reaction (MCR) gene drive, targeting the yellow gene of Drosophila melanogaster, replaced drive alleles with wild-type alleles demonstrating proof-of-principle. Although the ReMEDE system requires further research and development, the technology has a number of attractive features as a gene drive mitigation strategy, chief among these the potential to restore a wild-type population without releasing additional transgenic organisms or large-scale environmental engineering efforts.

Genetic Approaches for Controlling CRISPR-based Autonomous Homing Gene Drives.

CRISPR-based autonomous homing gene drives are a potentially transformative technology with the power to reduce the prevalence of, or even eliminate, vector-borne diseases, agricultural pests, and invasive species. However, there are a number of regulatory, ethical, environmental, and sociopolitical concerns surrounding the potential use of gene drives, particularly regarding the possibility for any unintended outcomes that might result from such a powerful technology. Therefore, there is an imminent need for countermeasures or technologies capable of exerting precise spatiotemporal control of gene drives, if their transformative potential is ever to be fully realized. This review summarizes the current state of the art in the development of technologies to prevent the uncontrolled spread of CRISPR-based autonomous homing gene drives.

Engineering a self-eliminating transgene in the yellow fever mosquito, Aedes aegypti.

Promising genetics-based approaches are being developed to reduce or prevent the transmission of mosquito-vectored diseases. Less clear is how such transgenes can be removed from the environment, a concern that is particularly relevant for highly invasive gene drive transgenes. Here, we lay the groundwork for a transgene removal system based on single-strand annealing (SSA), a eukaryotic DNA repair mechanism. An SSA-based rescuer strain (kmoRG ) was engineered to have direct repeat sequences (DRs) in the Aedes aegypti kynurenine 3-monooxygenase (kmo) gene flanking the intervening transgenic cargo genes, DsRED and EGFP. Targeted induction of DNA double-strand breaks (DSBs) in the DsRED transgene successfully triggered complete elimination of the entire cargo from the kmoRG strain, restoring the wild-type kmo gene, and thereby, normal eye pigmentation. Our work establishes the framework for strategies to remove transgene sequences during the evaluation and testing of modified strains for genetics-based mosquito control.

Alphavirus-derived small RNAs modulate pathogenesis in disease vector mosquitoes.

Mosquito-borne viruses cause significant levels of morbidity and mortality in humans and domesticated animals. Maintenance of mosquito-borne viruses in nature requires a biological transmission cycle that involves alternating virus replication in a susceptible vertebrate and mosquito host. Although the vertebrate infection is acute and often associated with disease, continual transmission of these viruses in nature depends on the establishment of a persistent, nonpathogenic infection in the mosquito vector. An antiviral RNAi response has been shown to limit the replication of RNA viruses in flies. However, the importance of the RNAi pathway as an antiviral defense in mammals is unclear. Differences in the immune responses of mammals and mosquitoes may explain why these viruses are not generally associated with pathology in the invertebrate host. We identified virus-derived small interfering RNAs (viRNAs), 21 nt in length, in Aedes aegypti infected with the mosquito-borne virus, Sindbis (SINV). viRNAs had an asymmetric distribution that spanned the length of the SINV genome. To determine the role of viRNAs in controlling pathogenic potential, mosquitoes were infected with recombinant alphaviruses expressing suppressors of RNA silencing. Decreased survival was observed in mosquitoes in which the accumulation of viRNAs was suppressed. These results suggest that an exogenous siRNA pathway is essential to the survival of mosquitoes infected with alphaviruses and, thus, the maintenance of these viruses in nature.

The ß2Tubulin, Rad50-ATPase and enolase cis-regulatory regions mediate male germline expression in Tribolium castaneum.

Genetics-based pest management processes, including the sterile insect technique, are an effective method for the control of some pest insects. However, current SIT methods are not directly transferable to many important pest insect species due to the lack of genetic sexing strains. Genome editing is revolutionizing the way we conduct genetics in insects, including in Tribolium castaneum, an important genetic model and agricultural pest. We identified orthologues of ß2Tubulin, Rad50-ATPase and enolase in T. castaneum. Using RT-PCR, we confirmed that these genes are predominantly expressed in the testis. PiggyBac-based transformation of T. castaneum cis-regulatory regions derived from Tc-ß2t, Tc-rad50 or Tc-eno resulted in EGFP expression specifically in the T. castaneum testis. Additionally, we determined that each of these regulatory regions regulates EGFP expression in different cell types of the male gonad. Cis-regulatory regions from Tc-ß2t produced EGFP expression throughout spermatogenesis and also in mature sperms; Tc-rad50 resulted in expression only in the haploid spermatid, while Tc-eno expressed EGFP in late spermatogenesis. In summary, the regulatory cis-regions characterized in this study are not only suited to study male gonadal function but could be used for development of transgenic sexing strains that produce one sex in pest control strategies.

Aedes aegypti dyspepsia encodes a novel member of the SLC16 family of transporters and is critical for reproductive fitness.

As a key vector for major arthropod-borne viruses (arboviruses) such as dengue, Zika and chikungunya, control of Aedes aegypti represents a major challenge in public health. Bloodmeal acquisition is necessary for the reproduction of vector mosquitoes and pathogen transmission. Blood contains potentially toxic amounts of iron while it provides nutrients for mosquito offspring; disruption of iron homeostasis in the mosquito may therefore lead to novel control strategies. We previously described a potential iron exporter in Ae. aegypti after a targeted functional screen of ZIP (zinc-regulated transporter/Iron-regulated transporter-like) and ZnT (zinc transporter) family genes. In this study, we performed an RNAseq-based screen in an Ae. aegypti cell line cultured under iron-deficient and iron-excess conditions. A subset of differentially expressed genes were analyzed via a cytosolic iron-sensitive dual-luciferase reporter assay with several gene candidates potentially involved in iron transport. In vivo gene silencing resulted in significant reduction of fecundity (egg number) and fertility (hatch rate) for one gene, termed dyspepsia. Silencing of dyspepsia reduced the induction of ferritin expression in the midgut and also resulted in delayed/impaired excretion and digestion. Further characterization of this gene, including a more direct confirmation of its substrate (iron or otherwise), could inform vector control strategies as well as to contribute to the field of metal biology.

A knockout screen of genes expressed specifically in Ae. aegypti pupae reveals a critical role for stretchin in mosquito flight.

Aedes aegypti is a critical vector for transmitting Zika, dengue, chikungunya, and yellow fever viruses to humans. Genetic strategies to limit mosquito survival based upon sex distortion or disruption of development may be valuable new tools to control Ae. aegypti populations. We identified six genes with expression limited to pupal development; osi8 and osi11 (Osiris protein family), CPRs and CPF (cuticle protein family), and stretchin (a muscle protein). Heritable CRISPR/Cas9-mediated gene knockout of these genes did not reveal any defects in pupal development. However, stretchin-null mutations (strnΔ35/Δ41) resulted in flightless mosquitoes with an abnormal open wing posture. The inability of adult strnΔ35/Δ41 mosquitoes to fly restricted their escape from aquatic rearing media following eclosion, and substantially reduced adult survival rates. Transgenic strains which contain the EGFP marker gene under the control of strn regulatory regions (0.8 kb, 1.4 kb, and 2.2 kb upstream, respectively), revealed the gene expression pattern of strn in muscle-like tissues in the thorax during late morphogenesis from L4 larvae to young adults. We demonstrated that Ae. aegypti pupae-specific strn is critical for adult mosquito flight capability and a key late-acting lethal target for mosquito-borne disease control.

Making gene drive biodegradable.

Gene drive systems have long been sought to modify mosquito populations and thus combat malaria and dengue. Powerful gene drive systems have been developed in laboratory experiments, but may never be used in practice unless they can be shown to be acceptable through rigorous field-based testing. Such testing is complicated by the anticipated difficulty in removing gene drive transgenes from nature. Here, we consider the inclusion of self-elimination mechanisms into the design of homing-based gene drive transgenes. This approach not only caused the excision of the gene drive transgene, but also generates a transgene-free allele resistant to further action by the gene drive. Strikingly, our models suggest that this mechanism, acting at a modest rate (10%) as part of a single-component system, would be sufficient to cause the rapid reversion of even the most robust homing-based gene drive transgenes, without the need for further remediation. Modelling also suggests that unlike gene drive transgenes themselves, self-eliminating transgene approaches are expected to tolerate substantial rates of failure. Thus, self-elimination technology may permit rigorous field-based testing of gene drives by establishing strict time limits on the existence of gene drive transgenes in nature, rendering them essentially biodegradable. This article is part of the theme issue 'Novel control strategies for mosquito-borne diseases'.