Search

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. CRISPR-based engineering can be used to bias sex ratios. Here the authors develop a transgenic line of Drosophila melanogaster expressing Cas9 from the Y chromosome and functionally characterize the utility of this strain for both sex selection and gene drive. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. Mosquito-borne diseases pose significant global health burdens. In this review, the authors explore Wolbachia and genome engineering approaches to mosquito-borne disease population control. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

With dengue virus (DENV) becoming endemic in tropical and subtropical regions worldwide, there is a pressing global demand for effective strategies to control the mosquitoes that spread this disease. Recent advances in genetic engineering technologies have made it possible to create mosquitoes with reduced vector competence, limiting their ability to acquire and transmit pathogens. Here we describe the development of Aedes aegypti mosquitoes synthetically engineered to impede vector competence to DENV. These mosquitoes express a gene encoding an engineered single-chain variable fragment derived from a broadly neutralizing DENV human monoclonal antibody and have significantly reduced viral infection, dissemination, and transmission rates for all four major antigenically distinct DENV serotypes. Importantly, this is the first engineered approach that targets all DENV serotypes, which is crucial for effective disease suppression. These results provide a compelling route for developing effective genetic-based DENV control strategies, which could be extended to curtail other arboviruses. Author summary: With limited success of traditional vector control methods to curb dengue infections and more than half of the world's population still at risk, there is a need for novel strategies to reduce its impact on public health. Recent advances in genetic technologies has allowed for precise modifications of mosquito genome to make them resistant to infections, thus breaking the transmission cycle. Here we generated engineered Ae. aegypti mosquitoes efficiently expressing a DENV-targeting single-chain variable fragment (scFv) derived from a previously characterized broadly neutralizing human antibody, which blocked infection and transmission in these mosquitoes. To our knowledge, this is the first example of an engineered transgene capable of rendering Ae. aegypti mosquitoes 100% refractory to all four serotypes of DENV. The engineered mosquitoes, in future, could easily be paired with a gene drive, capable of spreading the transgene throughout wild disease-transmitting mosquito populations and preventing further DENV transmission. Since a number of diverse and well-characterized antibodies exist against other arboviruses (eg chikungunya and Zika, this work also provides a proof-of-concept principle for developing similar genetic strategies for reducing the impact of these arboviruses. [ABSTRACT FROM AUTHOR]

Recent Zika virus (ZIKV) outbreaks have highlighted the necessity for development of novel vector control strategies to combat arboviral transmission, including genetic versions of the sterile insect technique, artificial infection with Wolbachia to reduce population size and/or vectoring competency, and gene drive-based methods. Here, we describe the development of mosquitoes synthetically engineered to impede vector competence to ZIKV. We demonstrate that a polycistronic cluster of engineered synthetic small RNAs targeting ZIKV is expressed and fully processed in Aedes aegypti, ensuring the formation of mature synthetic small RNAs in the midgut where ZIKV resides in the early stages of infection. Critically, we demonstrate that engineered Ae. aegypti mosquitoes harboring the anti-ZIKV transgene have significantly reduced viral infection, dissemination, and transmission rates of ZIKV. Taken together, these compelling results provide a promising path forward for development of effective genetic-based ZIKV control strategies, which could potentially be extended to curtail other arboviruses. [ABSTRACT FROM AUTHOR]

In Fig 1B, the columns corresponding to infection with DENV-3 and DENV-4 are duplicated and both denote the DENV-4 data. Reference 1 Buchman A, Gamez S, Li M, Antoshechkin I, Li H-H, Wang H-W, et al. (2020) Broad dengue neutralization in mosquitoes expressing an engineered antibody. [Extracted from the article]

Ongoing speciation in the most important African malaria vectors gives rise to cryptic populations, which differ remarkably in their behavior, ecology, and capacity to vector malaria parasites. Understanding the population structure and the drivers of genetic differentiation among mosquitoes is crucial for effective disease control because heterogeneity within vector species contributes to variability in malaria cases and allow fractions of populations to escape control efforts. To examine population structure and the potential impacts of recent large-scale control interventions, we have investigated the genomic patterns of differentiation in mosquitoes belonging to the Anopheles nili group-a large taxonomic group that diverged ~3 Myr ago. Using 4,343 single nucleotide polymorphisms (SNPs), we detected strong population structure characterized by high- FST values between multiple divergent populations adapted to different habitats within the Central African rainforest. Delineating the cryptic species within the Anopheles nili group is challenging due to incongruence between morphology, ribosomal DNA, and SNP markers consistent with incomplete lineage sorting and/or interspecific gene flow. A very high proportion of loci are fixed ( FST = 1) within the genome of putative species, which suggests that ecological and/or reproductive barriers are maintained by strong selection on a substantial number of genes. [ABSTRACT FROM AUTHOR]

The Anopheles gambiae complex contains a number of highly anthropophilic mosquito species that have acquired exceptional ability to thrive in complex human habitats. Thus, examining the evolutionary history of this Afrotropical mosquito may yield vital information on the selective processes that occurred during the adaptation to human-dominated environments. We performed reduced representation sequencing on 941 mosquitoes of the Anopheles gambiae complex collected across four ecogeographic zones in Cameroon. We find evidence for genetic and geographic subdivision within An. coluzzii and An. gambiae sensu stricto--the two most significant malaria vectors in the region. Importantly, in both species, rural and urban populations are genetically differentiated. Genome scans reveal pervasive signatures of selection centered on genes involved in xenobiotic resistance. Notably, a selective sweep containing detoxification enzymes is prominent in urban mosquitoes that exploit polluted breeding sites. Overall, our study suggests that recent anthropogenic environmental modifications and widespread use of insecticides are driving population differentiation and local adaptation in vectors with potentially significant consequences for malaria epidemiology. [ABSTRACT FROM AUTHOR]

Anopheles gambiae is amajor mosquito vector ofmalaria in Africa. Although increased use of insecticide-based vector control tools hasdecreased malaria transmission, elimination is likely torequire novelgenetic control strategies. It canbearguedthat theabsenceof an A. gambiae inbred line has slowed progress toward genetic vector control. In order to empower genetic studies and enable precise and reproducible experimentation, we set out to create an inbred line of this species. We found that amenability to inbreeding varied between populations of A. gambiae. After full-sib inbreeding for ten generations, we genotyped 112 individuals-56 saved prior to inbreeding and 56 collected after inbreeding-at a genome-wide panel of single nucleotide polymorphisms (SNPs). Although inbreeding dramatically reduced diversity across much of the genome, we discovered numerous, discrete genomic blocks that maintained high heterozygosity. For one large genomic region, we were able to definitively show that high diversity is due to the persistent polymorphismof a chromosomal inversion. Inbred lines in other eukaryotes often exhibit a qualitatively similar retention of polymorphism when typed at a small number ofmarkers. Our whole-genome SNP data provide the first strong, empirical evidence supporting associative overdominance as the mechanism maintaining higher than expected diversity in inbred lines. Although creation of A. gambiae lines devoid of nearly all polymorphism may not be feasible, our results provide critical insights into how more fully isogenic lines can be created. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]