Germline Cas9 expression yields highly efficient genome engineering in a major worldwide disease vector, Aedes aegypti.
Germline Cas9 expression yields highly efficient genome engineering in a major worldwide disease vector, Aedes aegypti.
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DOI：
10.1073/pnas.1711538114
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发表时间：
2017-12-05
影响因子：
11.1
通讯作者：
Akbari OS
中科院分区：
文献类型：
--
作者：
Li M;Bui M;Yang T;Bowman CS;White BJ;Akbari OS
埃及伊蚊是严重影响人类健康的多种虫媒病毒的主要载体，包括登革热、基孔肯雅热和寨卡病毒。在这种蚊子中开发有效的基因组工程工具不仅将为遗传控制策略的应用奠定基础，而且还将加速对疾病传播所涉及的关键生物过程的基础研究。在这里，我们报告了一种转基因CRISPR方法的发展，用于在这种生物体中快速破坏基因。鉴于其高编辑效率，我们开发的Cas9菌株可用于快速产生基因组修饰，允许高通量基因靶向，并可能促进基因驱动的开发，从而加速全面的功能注释和创新的种群控制策略的开发。CRISPR/Cas9技术的发展极大地提高了许多生物体中基因组编辑的可及性和效率。通常，Cas9的体内种系表达导致比胚胎注射显著更高的活性。然而，尚未开发出用于主要蚊子疾病载体埃及伊蚊的表达Cas9的转基因系。在这里，我们描述了多个稳定的，转基因Ae的一代。埃及菌株在种系中表达Cas9，导致使用CRISPR进行基因组修饰的一致性和效率的显著改善。使用这些菌株，我们破坏了许多对正常形态发育重要的基因，甚至从一次注射中产生了三重突变体。我们还设法将同源定向修复的速率提高了一个数量级以上。鉴于我们工程化的Cas9菌株的卓越诱变效率和特异性，它们可用于高通量反向遗传筛选，以帮助功能性地注释Ae。埃及人基因组此外，这些菌株代表了一个新的人口控制技术的发展目标Ae的一步。依赖Cas9基因驱动的埃及人。
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 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. 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 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.
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DOI：
10.1073/pnas.82.21.7369
发表时间：
1985-01-01
影响因子：
11.1
作者：
BIESSMANN, H
通讯作者：
BIESSMANN, H
DOI：
10.1126/science.1159689
发表时间：
2008-08-15
期刊：
Science (New York, N.Y.)
影响因子：
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作者：
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van der Oost J
影响因子：
3.7
作者：
Dong S;Lin J;Held NL;Clem RJ;Passarelli AL;Franz AW
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Franz AW
DOI：
10.1073/pnas.1521077112
发表时间：
2015-12-08
影响因子：
11.1
作者：
Gantz, Valentino M.;Jasinskiene, Nijole;James, Anthony A.
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影响因子：
4.8
作者：
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