Bioinformatic and cell-based tools for pooled CRISPR knockout screening in mosquitos.
Bioinformatic and cell-based tools for pooled CRISPR knockout screening in mosquitos.
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DOI：
10.1038/s41467-021-27129-3
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发表时间：
2021-11-24
影响因子：
16.6
通讯作者：
Perrimon N
中科院分区：
文献类型：
--
作者：
Viswanatha R;Mameli E;Rodiger J;Merckaert P;Feitosa-Suntheimer F;Colpitts TM;Mohr SE;Hu Y;Perrimon N
蚊媒疾病是世界范围内的公共卫生负担。目前理解和对抗它们的努力得到了利用培养的蚊子细胞的帮助。此外，在哺乳动物细胞中应用正向遗传方法，如CRISPR筛选，已经确定了寄主-病原体相互作用所需的必要基因和基因，通常有助于对基因的功能注释。目前还缺乏一种对蚊子细胞系进行遗传筛选的同等方法。为了开发这种方法，我们设计了一个新的生物信息学门户网站，用于在几种蚊子基因组中设计sgRNA文库，设计蚊子细胞系来表达Cas9并大规模接受sgRNA，并寻找在几种蚊子中表达sgRNA的最佳启动子。然后，我们优化了重组介导盒交换系统，以传递CRISPR sgRNA，并在按蚊细胞系中进行池CRISPR筛选。总之，我们为从疾病媒介物种的细胞系中进行高通量基因组规模筛选提供了一个平台。正向遗传方法，如CRISPR筛查，是识别基本基因和影响宿主-病原体相互作用的基因的有效方法。在这里，作者设计了一个用于sgRNA设计的生物信息学门户和一个用于运送到蚊子细胞系中的重组介导盒系统。
Mosquito-borne diseases present a worldwide public health burden. Current efforts to understand and counteract them have been aided by the use of cultured mosquito cells. Moreover, application in mammalian cells of forward genetic approaches such as CRISPR screens have identified essential genes and genes required for host-pathogen interactions, and in general, aided in functional annotation of genes. An equivalent approach for genetic screening of mosquito cell lines has been lacking. To develop such an approach, we design a new bioinformatic portal for sgRNA library design in several mosquito genomes, engineer mosquito cell lines to express Cas9 and accept sgRNA at scale, and identify optimal promoters for sgRNA expression in several mosquito species. We then optimize a recombination-mediated cassette exchange system to deliver CRISPR sgRNA and perform pooled CRISPR screens in an Anopheles cell line. Altogether, we provide a platform for high-throughput genome-scale screening in cell lines from disease vector species. Forward genetic approaches such as CRISPR screens are powerful ways to identify essential genes and those that influence host-pathogen interactions. Here the authors design a bioinformatics portal for sgRNA design and a recombination-mediated cassette system for delivery into mosquito cell lines.
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影响因子：
7.3
作者：
Housden BE;Valvezan AJ;Kelley C;Sopko R;Hu Y;Roesel C;Lin S;Buckner M;Tao R;Yilmazel B;Mohr SE;Manning BD;Perrimon N
通讯作者：
Perrimon N
影响因子：
5.6
作者：
Ford K;McDonald D;Mali P
通讯作者：
Mali P
影响因子：
3.8
作者：
Azlan A;Obeidat SM;Theva Das K;Yunus MA;Azzam G
通讯作者：
Azzam G
影响因子：
46.9
作者：
Doench JG;Fusi N;Sullender M;Hegde M;Vaimberg EW;Donovan KF;Smith I;Tothova Z;Wilen C;Orchard R;Virgin HW;Listgarten J;Root DE
通讯作者：
Root DE
影响因子：
2.1
作者：
HSU, SH;MAO, WH;CROSS, JH
通讯作者：
CROSS, JH