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

Ming Li, Michelle Bui, Ting Yang, Bradley J. White, Omar S. Akbari
doi: https://doi.org/10.1101/156778
Ming Li
1Department of Entomology and Center for Disease Vector Research, Institute for Integrative Genome Biology, University of California, Riverside, Riverside, CA 92521, USA
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Michelle Bui
1Department of Entomology and Center for Disease Vector Research, Institute for Integrative Genome Biology, University of California, Riverside, Riverside, CA 92521, USA
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Ting Yang
1Department of Entomology and Center for Disease Vector Research, Institute for Integrative Genome Biology, University of California, Riverside, Riverside, CA 92521, USA
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Bradley J. White
1Department of Entomology and Center for Disease Vector Research, Institute for Integrative Genome Biology, University of California, Riverside, Riverside, CA 92521, USA
2Current address: Verily Life Sciences, 259 East Grand Avenue, South San Francisco, CA
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Omar S. Akbari
1Department of Entomology and Center for Disease Vector Research, Institute for Integrative Genome Biology, University of California, Riverside, Riverside, CA 92521, USA
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  • For correspondence: oakbari@ucr.edu
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Abstract

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.

Significance Statement Aedes 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.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted June 27, 2017.
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Germline Cas9 Expression Yields Highly Efficient Genome Engineering in a Major Worldwide Disease Vector, Aedes aegypti
Ming Li, Michelle Bui, Ting Yang, Bradley J. White, Omar S. Akbari
bioRxiv 156778; doi: https://doi.org/10.1101/156778
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Germline Cas9 Expression Yields Highly Efficient Genome Engineering in a Major Worldwide Disease Vector, Aedes aegypti
Ming Li, Michelle Bui, Ting Yang, Bradley J. White, Omar S. Akbari
bioRxiv 156778; doi: https://doi.org/10.1101/156778

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