Evaluation and Design of Genome-Wide CRISPR/SpCas9 Knockout Screens.
Evaluation and Design of Genome-Wide CRISPR/SpCas9 Knockout Screens.
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DOI：
10.1534/g3.117.041277
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发表时间：
2017-08-07
期刊：
影响因子：
--
通讯作者：
Moffat J
中科院分区：
文献类型：
--
作者：
Hart T;Tong AHY;Chan K;Van Leeuwen J;Seetharaman A;Aregger M;Chandrashekhar M;Hustedt N;Seth S;Noonan A;Habsid A;Sizova O;Nedyalkova L;Climie R;Tworzyanski L;Lawson K;Sartori MA;Alibeh S;Tieu D;Masud S;Mero P;Weiss A;Brown KR;Usaj M;Billmann M;Rahman M;Constanzo M;Myers CL;Andrews BJ;Boone C;Durocher D;Moffat J
CRISPR/SpCas9 技术对哺乳动物细胞系的适应正在改变人类功能基因组学的研究。针对人类蛋白质编码基因并在病毒载体中编码的 CRISPR 引导 RNA (gRNA) 混合文库已用于在多种人类癌症和永生化细胞系中系统地创建基因敲除，以鉴定这些敲除是否会导致细胞健康缺陷。之前的工作表明，在类似的检测中，CRISPR 筛选比混合文库 shRNA 筛选更灵敏、更特异，但目前 CRISPR 文库设计和实验方案之间存在显着差异。在这项研究中，我们使用三个不同的基因组规模 gRNA 文库重新分析了来自三个研究小组的 17 个人类细胞系基因组规模敲除筛选。使用基因必需性贝叶斯分析算法来识别必需基因，我们将之前定义的人类核心必需基因集从 360 个基因细化并扩展为 684 个基因。我们使用这套扩展的参考核心必需基因 CEG2 以及来自六个 CRISPR 敲除筛选的经验数据来指导序列优化的 gRNA 文库，即 Toronto KnockOut 3.0 版 (TKOv3) 文库的设计。然后，我们证明了该文库相对于必需和非必需基因的参考集以及使用类似方法的其他筛选的高效性。优化的 TKOv3 文库与 CEG2 参考集相结合，为在人类细胞系中执行和评估基因敲除筛选提供了一个高效、高度优化的平台。
The adaptation of CRISPR/SpCas9 technology to mammalian cell lines is transforming the study of human functional genomics. Pooled libraries of CRISPR guide RNAs (gRNAs) targeting human protein-coding genes and encoded in viral vectors have been used to systematically create gene knockouts in a variety of human cancer and immortalized cell lines, in an effort to identify whether these knockouts cause cellular fitness defects. Previous work has shown that CRISPR screens are more sensitive and specific than pooled-library shRNA screens in similar assays, but currently there exists significant variability across CRISPR library designs and experimental protocols. In this study, we reanalyze 17 genome-scale knockout screens in human cell lines from three research groups, using three different genome-scale gRNA libraries. Using the Bayesian Analysis of Gene Essentiality algorithm to identify essential genes, we refine and expand our previously defined set of human core essential genes from 360 to 684 genes. We use this expanded set of reference core essential genes, CEG2, plus empirical data from six CRISPR knockout screens to guide the design of a sequence-optimized gRNA library, the Toronto KnockOut version 3.0 (TKOv3) library. We then demonstrate the high effectiveness of the library relative to reference sets of essential and nonessential genes, as well as other screens using similar approaches. The optimized TKOv3 library, combined with the CEG2 reference set, provide an efficient, highly optimized platform for performing and assessing gene knockout screens in human cell lines.
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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：
8.8
作者：
Tzelepis K;Koike-Yusa H;De Braekeleer E;Li Y;Metzakopian E;Dovey OM;Mupo A;Grinkevich V;Li M;Mazan M;Gozdecka M;Ohnishi S;Cooper J;Patel M;McKerrell T;Chen B;Domingues AF;Gallipoli P;Teichmann S;Ponstingl H;McDermott U;Saez-Rodriguez J;Huntly BJP;Iorio F;Pina C;Vassiliou GS;Yusa K
通讯作者：
Yusa K
影响因子：
56.9
作者：
Blomen, Vincent A.;Majek, Peter;Brummelkamp, Thijn R.
通讯作者：
Brummelkamp, Thijn R.
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
影响因子：
46.9
作者：
Evers, Bastiaan;Jastrzebski, Katarzyna;Bernards, Rene
通讯作者：
Bernards, Rene