Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities
Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities
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DOI：
10.1038/s41467-018-07901-8
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发表时间：
2018-12-21
影响因子：
16.6
通讯作者：
Doench, John G.
中科院分区：
文献类型：
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作者：
Sanson, Kendall R.;Hanna, Ruth E.;Doench, John G.
用于CRISPR敲除（CRISPRko）、干扰（CRISPRi）和激活（CRISPRa）的全基因组文库的创建使得能够系统地询问基因功能。在这里，我们表明，我们最近描述的CRISPRko文库（Brunello）在区分必需和非必需基因方面比以前发表的文库更有效，提供了与GeCKO通过RNAi提供的GeCKO文库大致相同的扰动水平性能改善。此外，我们提出了CRISPRi（Dolcetto）和CRISPRa（Calabrese）的全基因组文库，并在阴性选择筛选中显示，Dolcetto每个基因的sgRNA较少，优于现有的CRISPRi文库，并在检测必需基因方面达到了与CRISPRko相当的性能。我们还进行了阳性选择CRISPRa筛选，并证明Calabrese在鉴定vemurafenib抗性基因方面优于SAM方法。我们进一步将CRISPRa与开放阅读框（ORF）的基因组规模文库进行比较。总之，这些文库代表了一套全基因组工具，可以用多种方式有效地询问基因功能。
The creation of genome-wide libraries for CRISPR knockout (CRISPRko), interference (CRISPRi), and activation (CRISPRa) has enabled the systematic interrogation of gene function. Here, we show that our recently-described CRISPRko library (Brunello) is more effective than previously published libraries at distinguishing essential and non-essential genes, providing approximately the same perturbation-level performance improvement over GeCKO libraries as GeCKO provided over RNAi. Additionally, we present genome-wide libraries for CRISPRi (Dolcetto) and CRISPRa (Calabrese), and show in negative selection screens that Dolcetto, with fewer sgRNAs per gene, outperforms existing CRISPRi libraries and achieves comparable performance to CRISPRko in detecting essential genes. We also perform positive selection CRISPRa screens and demonstrate that Calabrese outperforms the SAM approach at identifying vemurafenib resistance genes. We further compare CRISPRa to genome-scale libraries of open reading frames (ORFs). Together, these libraries represent a suite of genome-wide tools to efficiently interrogate gene function with multiple modalities.