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bioRxiv2017

Highly efficient site-specific mutagenesis in Malaria mosquitoes using CRISPR

Anopheles mosquitoes transmit at least 200 million annual malaria infections worldwide. Despite considerable genomic resources, mechanistic understanding of biological processes in Anopheles has been hampered by a lack of tools for reverse genetics. Here, we report successful application of the CRISPR/Cas9 system for highly efficient, site-specific mutagenesis in the diverse malaria vectors Anopheles albimanus, Anopheles coluzzii, and Anopheles funestus. When guide RNAs and Cas9 are injected at high concentration, germline mutations are common and usually bi-allelic allowing for the rapid creation of stable, mutant lines for reverse genetic analysis. Our protocol should enable researchers to dissect the molecular and cellular basis of anopheline traits critical to successful disease transmission, potentially exposing new targets for malaria control.

genetics↗

bioRxiv2017

Germline Cas9 Expression Yields Highly Efficient Genome Engineering in a Major Worldwide Disease Vector, Aedes aegypti

The development of CRISPR/Cas9 technologies has dramatically increased the accessibility and efficiency of genome editing in many organisms. In general, in vivo germline expression of Cas9 results in substantially higher activity than embryonic injection. However, no transgenic lines expressing Cas9 have been developed for the major mosquito disease vector Aedes aegypti. Here, we describe the generation of multiple stable, transgenic Ae. aegypti strains expressing Cas9 in the germline, resulting in dramatic improvements in both the consistency and efficiency of genome modifications using CRISPR. Using these strains, we disrupted numerous genes important for normal morphological development, and even generated triple mutants from a single injection. We have also managed to increase the rates of homology directed repair by more than an order of magnitude. Given the exceptional mutagenic efficiency and specificity of the Cas9 strains we built, they can be used for high-throughput reverse genetic screens to help functionally annotate the Ae. aegypti genome. Additionally, these strains represent a first step towards the development of novel population control technologies targeting Ae. aegypti that rely on Cas9-based gene drives.\n\nSignificance StatementAedes aegypti is the principal vector of multiple arboviruses that significantly affect human health including dengue, chikungunya, and zika. Development of tools for efficient genome engineering in this mosquito will not only lay the foundation for the application of novel genetic control strategies that do not rely on insecticides, but will also accelerate basic research on key biological processes involved in disease transmission. Here, we report the development of a transgenic CRISPR approach for rapid gene disruption in this organism. Given their high editing efficiencies, the Cas9 strains we developed can be used to quickly generate novel genome modifications allowing for high-throughput gene targeting, and can possibly facilitate the development of gene drives, thereby accelerating comprehensive functional annotation and development of innovative population control strategies for Ae. aegypti.

bioengineering↗