Enhancing the genome editing toolbox: genome wide CRISPR arrayed libraries.
Enhancing the genome editing toolbox: genome wide CRISPR arrayed libraries.
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DOI：
10.1038/s41598-017-01766-5
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发表时间：
2017-05-22
影响因子：
4.6
通讯作者：
Bradley A
中科院分区：
文献类型：
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作者：
Metzakopian E;Strong A;Iyer V;Hodgkins A;Tzelepis K;Antunes L;Friedrich MJ;Kang Q;Davidson T;Lamberth J;Hoffmann C;Davis GD;Vassiliou GS;Skarnes WC;Bradley A
CRISPR-Cas9技术加速了生物学研究成为许多实验室的常规。它正在迅速取代传统的基因编辑技术，并在全基因组和基因聚焦应用中具有很高的实用性。在这里，我们展示了第一个单独克隆的CRISPR-Cas9全基因组阵列sgRNA文库，涵盖17，166个人类和20，430个小鼠基因，人类基因组的复杂性为34，332个sgRNA，小鼠基因组的复杂性为40，860个sgRNA。为了灵活地产生稳定的细胞系，已将sgRNA克隆到含有PiggyBac转座酶识别元件以及荧光和药物选择标记的慢病毒骨架中。如通过CEL-1测定所测量的，超过95%的测试的sgRNA诱导特异性DNA切割。此外，靶向GPI锚蛋白途径基因的sgRNA诱导通过FLAER标记测量的人和小鼠细胞系中的功能丧失突变。这些阵列文库提供了对单个基因、组合以及更大的基因集进行筛选的前景。它们还促进了来自全基因组筛选的信号的快速去卷积。这组载体提供了一个有组织的综合基因编辑工具箱，具有相当大的科学价值。
CRISPR-Cas9 technology has accelerated biological research becoming routine for many laboratories. It is rapidly replacing conventional gene editing techniques and has high utility for both genome-wide and gene-focussed applications. Here we present the first individually cloned CRISPR-Cas9 genome wide arrayed sgRNA libraries covering 17,166 human and 20,430 mouse genes at a complexity of 34,332 sgRNAs for human and 40,860 sgRNAs for the mouse genome. For flexibility in generating stable cell lines the sgRNAs have been cloned in a lentivirus backbone containing PiggyBac transposase recognition elements together with fluorescent and drug selection markers. Over 95% of tested sgRNA induced specific DNA cleavage as measured by CEL-1 assays. Furthermore, sgRNA targeting GPI anchor protein pathway genes induced loss of function mutations in human and mouse cell lines measured by FLAER labelling. These arrayed libraries offer the prospect for performing screens on individual genes, combinations as well as larger gene sets. They also facilitate rapid deconvolution of signals from genome-wide screens. This set of vectors provide an organized comprehensive gene editing toolbox of considerable scientific value.