A multiplexed, confinable CRISPR/Cas9 gene drive can propagate in caged Aedes aegypti populations.

Aedes aegypti is the main vector of several major pathogens including dengue, Zika and chikungunya viruses. Classical mosquito control strategies utilizing insecticides are threatened by rising resistance. This has stimulated interest in new genetic systems such as gene drivesHere, we test the regulatory sequences from the Ae. aegypti benign gonial cell neoplasm (bgcn) homolog to express Cas9 and a separate multiplexing sgRNA-expressing cassette inserted into the Ae. aegypti kynurenine 3-monooxygenase (kmo) gene. When combined, these two elements provide highly effective germline cutting at the kmo locus and act as a gene drive. Our target genetic element drives through a cage trial population such that carrier frequency of the element increases from 50% to up to 89% of the population despite significant fitness costs to kmo insertions. Deep sequencing suggests that the multiplexing design could mitigate resistance allele formation in our gene drive system.

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.

Engineered Antiviral Sensor Targets Infected Mosquitoes.

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.

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.

AePUb promoter length modulates gene expression in Aedes aegypti.

Molecular tools for modulating transgene expression in Aedes aegypti are few. Here we demonstrate that adjustments to the AePUb promoter length can alter expression levels of two reporter proteins in Ae. aegypti cell culture and in mosquitoes. This provides a simple means for increasing or decreasing expression of a gene of interest and easy translation from cells to whole insects.

Optimizing CRE and PhiC31 mediated recombination in Aedes aegypti.

Introduction: Genetic manipulation of Aedes aegypti is key to developing a deeper understanding of this insects' biology, vector-virus interactions and makes future genetic control strategies possible. Despite some advances, this process remains laborious and requires highly skilled researchers and specialist equipment. Methods: Here we present two improved methods for genetic manipulation in this species. Use of transgenic lines which express Cre recombinase and a plasmid-based method for expressing PhiC31 when injected into early embryos. Results: Use of transgenic lines which express Cre recombinase allowed, by simple crossing schemes, germline or somatic recombination of transgenes, which could be utilized for numerous genetic manipulations. PhiC31 integrase based methods for site-specific integration of genetic elements was also improved, by developing a plasmid which expresses PhiC31 when injected into early embryos, eliminating the need to use costly and unstable mRNA as is the current standard. Discussion: Here we have expanded the toolbox for synthetic biology in Ae. aegypti. These methods can be easily transferred into other mosquito and even insect species by identifying appropriate promoter sequences. This advances the ability to manipulate these insects for fundamental studies, and for more applied approaches for pest control.

Mimicking superinfection exclusion disrupts alphavirus infection and transmission in the yellow fever mosquito Aedes aegypti.

Multiple viruses, including pathogenic viruses, bacteriophages, and even plant viruses, cause a phenomenon termed superinfection exclusion whereby a currently infected cell is resistant to secondary infection by the same or a closely related virus. In alphaviruses, this process is thought to be mediated, at least in part, by the viral protease (nsP2) which is responsible for processing the nonstructural polyproteins (P123 and P1234) into individual proteins (nsP1-nsP4), forming the viral replication complex. Taking a synthetic biology approach, we mimicked this naturally occurring phenomenon by generating a superinfection exclusion-like state in Aedes aegypti mosquitoes, rendering them refractory to alphavirus infection. By artificially expressing Sindbis virus (SINV) and chikungunya virus (CHIKV) nsP2 in mosquito cells and transgenic mosquitoes, we demonstrated a reduction in both SINV and CHIKV viral replication rates in cells following viral infection as well as reduced infection prevalence, viral titers, and transmission potential in 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.

Susceptibility of Aedes albopictus, Ae. aegypti and human populations to Ross River virus in Kuala Lumpur, Malaysia.

BACKGROUND: Emerging arboviruses such as chikungunya and Zika viruses have unexpectedly caused widespread outbreaks in tropical and subtropical regions recently. Ross River virus (RRV) is endemic in Australia and has epidemic potential. In Malaysia, Aedes mosquitoes are abundant and drive dengue and chikungunya outbreaks. We assessed risk of an RRV outbreak in Kuala Lumpur, Malaysia by determining vector competence of local Aedes mosquitoes and local seroprevalence as a proxy of human population susceptibility. METHODOLOGY/PRINCIPAL FINDINGS: We assessed oral susceptibility of Malaysian Ae. aegypti and Ae. albopictus by real-time PCR to an Australian RRV strain SW2089. Replication kinetics in midgut, head and saliva were determined at 3 and 10 days post-infection (dpi). With a 3 log10 PFU/ml blood meal, infection rate was higher in Ae. albopictus (60%) than Ae. aegypti (15%; p<0.05). Despite similar infection rates at 5 and 7 log10 PFU/ml blood meals, Ae. albopictus had significantly higher viral loads and required a significantly lower median oral infectious dose (2.7 log10 PFU/ml) than Ae. aegypti (4.2 log10 PFU/ml). Ae. albopictus showed higher vector competence, with higher viral loads in heads and saliva, and higher transmission rate (RRV present in saliva) of 100% at 10 dpi, than Ae. aegypti (41%). Ae. aegypti demonstrated greater barriers at either midgut escape or salivary gland infection, and salivary gland escape. We then assessed seropositivity against RRV among 240 Kuala Lumpur inpatients using plaque reduction neutralization, and found a low rate of 0.8%. CONCLUSIONS/SIGNIFICANCE: Both Ae. aegypti and Ae. albopictus are susceptible to RRV, but Ae. albopictus displays greater vector competence. Extensive travel links with Australia, abundant Aedes vectors, and low population immunity places Kuala Lumpur, Malaysia at risk of an imported RRV outbreak. Surveillance and increased diagnostic awareness and capacity are imperative to prevent establishment of new arboviruses in Malaysia.

Engineered Antiviral Sensor Targets Infected Mosquitoes.

Escalating vector disease burdens pose significant global health risks, so innovative tools for targeting mosquitoes are critical. 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. Akin to a stealthy Trojan Horse hiding in stealth awaiting the presence of its enemy, REAPER remains concealed within the mosquito until an infectious blood meal is up taken. 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 its promiscuous collateral activity can even kill infected mosquitoes. This innovative REAPER technology adds to an arsenal of effective molecular genetic tools to combat mosquito virus transmission.

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.

A CRISPR endonuclease gene drive reveals distinct mechanisms of inheritance bias.

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.

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.

Daisy-chain gene drives: The role of low cut-rate, resistance mutations, and maternal deposition.

The introgression of genetic traits through gene drive may serve as a powerful and widely applicable method of biological control. However, for many applications, a self-perpetuating gene drive that can spread beyond the specific target population may be undesirable and preclude use. Daisy-chain gene drives have been proposed as a means of tuning the invasiveness of a gene drive, allowing it to spread efficiently into the target population, but be self-limiting beyond that. Daisy-chain gene drives are made up of multiple independent drive elements, where each element, except one, biases the inheritance of another, forming a chain. Under ideal inheritance biasing conditions, the released drive elements remain linked in the same configuration, generating copies of most of their elements except for the last remaining link in the chain. Through mathematical modelling of populations connected by migration, we have evaluated the effect of resistance alleles, different fitness costs, reduction in the cut-rate, and maternal deposition on two alternative daisy-chain gene drive designs. We find that the self-limiting nature of daisy-chain gene drives makes their spread highly dependent on the efficiency and fidelity of the inheritance biasing mechanism. In particular, reductions in the cut-rate and the formation of non-lethal resistance alleles can cause drive elements to lose their linked configuration. This severely reduces the invasiveness of the drives and allows for phantom cutting, where an upstream drive element cuts a downstream target locus despite the corresponding drive element being absent, creating and biasing the inheritance of additional resistance alleles. This phantom cutting can be mitigated by an alternative indirect daisy-chain design. We further find that while dominant fitness costs and maternal deposition reduce daisy-chain invasiveness, if overcome with an increased release frequency, they can reduce the spread of the drive into a neighbouring population.

The Acquisition and Retention of Lumpy Skin Disease Virus by Blood-Feeding Insects Is Influenced by the Source of Virus, the Insect Body Part, and the Time since Feeding.

Lumpy skin disease virus (LSDV) is a poxvirus that causes severe systemic disease in cattle and is spread by mechanical arthropod-borne transmission. This study quantified the acquisition and retention of LSDV by four species of Diptera (Stomoxys calcitrans, Aedes aegypti, Culex quinquefasciatus, and Culicoides nubeculosus) from cutaneous lesions, normal skin, and blood from a clinically affected animal. The acquisition and retention of LSDV by Ae. aegypti from an artificial membrane feeding system was also examined. Mathematical models of the data were generated to identify the parameters which influence insect acquisition and retention of LSDV. For all four insect species, the probability of acquiring LSDV was substantially greater when feeding on a lesion compared with feeding on normal skin or blood from a clinically affected animal. After feeding on a skin lesion LSDV was retained on the proboscis for a similar length of time (around 9 days) for all four species and for a shorter time in the rest of the body, ranging from 2.2 to 6.4 days. Acquisition and retention of LSDV by Ae. aegypti after feeding on an artificial membrane feeding system that contained a high titer of LSDV was comparable to feeding on a skin lesion on a clinically affected animal, supporting the use of this laboratory model as a replacement for some animal studies. This work reveals that the cutaneous lesions of LSD provide the high-titer source required for acquisition of the virus by insects, thereby enabling the mechanical vector-borne transmission. IMPORTANCE Lumpy skin disease virus (LSDV) is a high consequence pathogen of cattle that is rapidly expanding its geographical boundaries into new regions such as Europe and Asia. This expansion is promoted by the mechanical transmission of the virus via hematogenous arthropods. This study quantifies the acquisition and retention of LSDV by four species of blood-feeding insects and reveals that the cutaneous lesions of LSD provide the high titer virus source necessary for virus acquisition by the insects. An artificial membrane feeding system containing a high titer of LSDV was shown to be comparable to a skin lesion on a clinically affected animal when used as a virus source. This promotes the use of these laboratory-based systems as replacements for some animal studies. Overall, this work advances our understanding of the mechanical vector-borne transmission of LSDV and provides evidence to support the design of more effective disease control programmes.

Intron-derived small RNAs for silencing viral RNAs in mosquito cells.

Aedes aegypti and Ae. albopictus are the main vectors of mosquito-borne viruses of medical and veterinary significance. Many of these viruses have RNA genomes. Exogenously provided, e.g. transgene encoded, small RNAs could be used to inhibit virus replication, breaking the transmission cycle. We tested, in Ae. aegypti and Ae. albopictus cell lines, reporter-based strategies for assessing the ability of two types of small RNAs to inhibit a chikungunya virus (CHIKV) derived target. Both types of small RNAs use a Drosophila melanogaster pre-miRNA-1 based hairpin for their expression, either with perfect base-pairing in the stem region (shRNA-like) or containing two mismatches (miRNA-like). The pre-miRNA-1 stem loop structure was encoded within an intron; this allows co-expression of one or more proteins, e.g. a fluorescent protein marker tracking the temporal and spatial expression of the small RNAs in vivo. Three reporter-based systems were used to assess the relative silencing efficiency of ten shRNA-like siRNAs and corresponding miRNA-like designs. Two systems used a luciferase reporter RNA with CHIKV RNA inserted either in the coding sequence or within the 3' UTR. A third reporter used a CHIKV derived split replication system. All three reporters demonstrated that while silencing could be achieved with both miRNA-like and shRNA-like designs, the latter were substantially more effective. Dcr-2 was required for the shRNA-like siRNAs as demonstrated by loss of inhibition of the reporters in Dcr-2 deficient cell lines. These positive results in cell culture are encouraging for the potential use of this pre-miRNA-1-based system in transgenic mosquitoes.

Expression of Alphavirus Nonstructural Protein 2 (nsP2) in Mosquito Cells Inhibits Viral RNA Replication in Both a Protease Activity-Dependent and -Independent Manner.

Alphaviruses are positive-strand RNA viruses, mostly being mosquito-transmitted. Cells infected by an alphavirus become resistant to superinfection due to a block that occurs at the level of RNA replication. Alphavirus replication proteins, called nsP1-4, are produced from nonstructural polyprotein precursors, processed by the protease activity of nsP2. Trans-replicase systems and replicon vectors were used to study effects of nsP2 of chikungunya virus and Sindbis virus on alphavirus RNA replication in mosquito cells. Co-expressed wild-type nsP2 reduced RNA replicase activity of homologous virus; this effect was reduced but typically not abolished by mutation in the protease active site of nsP2. Mutations in the replicase polyprotein that blocked its cleavage by nsP2 reduced the negative effect of nsP2 co-expression, confirming that nsP2-mediated inhibition of RNA replicase activity is largely due to nsP2-mediated processing of the nonstructural polyprotein. Co-expression of nsP2 also suppressed the activity of replicases of heterologous alphaviruses. Thus, the presence of nsP2 inhibits formation and activity of alphavirus RNA replicase in protease activity-dependent and -independent manners. This knowledge improves our understanding about mechanisms of superinfection exclusion for alphaviruses and may aid the development of anti-alphavirus approaches.

Reproductive interference and Satyrisation: mechanisms, outcomes and potential use for insect control.

Reproductive Interference occurs when interactions between individuals from different species disrupt reproductive processes, resulting in a fitness cost to one or both parties involved. It is typically observed between individuals of closely related species, often upon secondary contact. In both vertebrates and invertebrates, Reproductive Interference is frequently referred to as 'Satyrisation'. It can manifest in various ways, ranging from blocking or reducing the efficacy of mating signals, through to negative effects of heterospecific copulations and the production of sterile or infertile hybrid offspring. The negative fitness effects of Satyrisation in reciprocal matings between species are often asymmetric and it is this aspect, which is most relevant to, and can offer utility in, pest management. In this review, we focus on Satyrisation and outline the mechanisms through which it can operate. We illustrate this by using test cases, and we consider the underlying reasons why the reproductive interactions that comprise Satyrisation occur. We synthesise the key factors affecting the expression of Satyrisation and explore how they have potential utility in developing new routes for the management and control of harmful insects. We consider how Satyrisation might interact with other control mechanisms, and conclude by outlining a framework for its use in control, highlighting some of the important next steps.

The Challenges in Developing Efficient and Robust Synthetic Homing Endonuclease Gene Drives.

Making discrete and precise genetic changes to wild populations has been proposed as a means of addressing some of the world's most pressing ecological and public health challenges caused by insect pests. Technologies that would allow this, such as synthetic gene drives, have been under development for many decades. Recently, a new generation of programmable nucleases has dramatically accelerated technological development. CRISPR-Cas9 has improved the efficiency of genetic engineering and has been used as the principal effector nuclease in different gene drive inheritance biasing mechanisms. Of these nuclease-based gene drives, homing endonuclease gene drives have been the subject of the bulk of research efforts (particularly in insects), with many different iterations having been developed upon similar core designs. We chart the history of homing gene drive development, highlighting the emergence of challenges such as unintended repair outcomes, "leaky" expression, and parental deposition. We conclude by discussing the progress made in developing strategies to increase the efficiency of homing endonuclease gene drives and mitigate or prevent unintended outcomes.