Germline Cas9 expression yields highly efficient genome engineering in a major worldwide disease vector, Aedes aegypti
Thursday, 2017/12/07 | 08:02:11
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Ming Li, Michelle Bui, Ting Yang, Christian S. Bowman, Bradley J. White and Omar S. Akbari PNAS December 5 2017; vol.114; no.49: E10540–E10549 GENETICS SignificanceAedes 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 genetic control strategies, 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 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. AbstractThe 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 engineered, they can be used for high-throughput reverse genetic screens to help functionally annotate the Ae. aegypti genome. Additionally, these strains represent a step toward the development of novel population control technologies targeting Ae. aegypti that rely on Cas9-based gene drives.
See: http://www.pnas.org/content/114/49/E10540.full
Figure 3: Single injections of multiplexed sgRNAa robustly generate double- and triple-mutant mosquitoes. Larva, pupae, and adult G1 phenotypes for double-mutants, including: yellow body and white eyes (yellow/white), a mixture of yellow and dark body (yellow/ebony), dark body and white eyes (ebony/white), and one triple-mutant, which is a phenotypic mixture of yellow and dark body and white eyes (yellow/ebony/white). The striking differences between wild-type and mutant larva, pupae and adult are highlighted. (Magnifications: whole-body images, 20×; Insets, 100×.) |
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