Evaluation of RNAi and CRISPR technologies by large-scale gene expression profiling in the Connectivity Map
Evaluation of RNAi and CRISPR technologies by large-scale gene expression profiling in the Connectivity Map
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DOI：
10.1101/147504
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发表时间：
2017-06
期刊：
影响因子：
9.8
通讯作者：
Ian Smith;Peyton Greenside;Ted E. Natoli;D. L. Lahr;David Wadden;I. Tirosh;Rajiv Narayan;D. Root;T. Golub;A. Subramanian;John G Doench
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文献类型：
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作者：
Ian Smith;Peyton Greenside;Ted E. Natoli;D. L. Lahr;David Wadden;I. Tirosh;Rajiv Narayan;D. Root;T. Golub;A. Subramanian;John G Doench
RNA干扰（RNAi）在哺乳动物细胞中的应用提供了进行表型筛选以确定基因功能的手段。尽管RNAi已经彻底改变了功能丧失遗传实验，但很难系统地评估脱靶效应的流行程度和后果。连接图（CMAP）代表了一种前所未有的资源，可用于研究表达短发夹RNA（shRNA）的基因表达后果。对9种细胞系中应用的超过13，000种shRNA的特征分析显示，RNAi的miRNA样脱靶效应比通常认识的要强得多，也更普遍。我们表明，减轻脱靶效应是可行的，在这些数据集通过计算方法产生一个共识基因签名（CGS）。此外，我们通过分析6个细胞系中的373个sgRNA，将RNAi技术与基于规则间隔短回文重复序列（CRISPR）的基因敲除技术进行了比较，结果表明，靶向功效相当，但CRISPR技术对系统性脱靶效应的敏感性要低得多。这些结果将有助于指导正确使用和分析的功能丧失试剂的基因功能的测定。
The application of RNA interference (RNAi) to mammalian cells has provided the means to perform phenotypic screens to determine the functions of genes. Although RNAi has revolutionized loss of function genetic experiments, it has been difficult to systematically assess the prevalence and consequences of off-target effects. The Connectivity Map (CMAP) represents an unprecedented resource to study the gene expression consequences of expressing short hairpin RNAs (shRNAs). Analysis of signatures for over 13,000 shRNAs applied in 9 cell lines revealed that miRNA-like off-target effects of RNAi are far stronger and more pervasive than generally appreciated. We show that mitigating off-target effects is feasible in these datasets via computational methodologies to produce a Consensus Gene Signature (CGS). In addition, we compared RNAi technology to clustered regularly interspaced short palindromic repeat (CRISPR)-based knockout by analysis of 373 sgRNAs in 6 cells lines, and show that the on-target efficacies are comparable, but CRISPR technology is far less susceptible to systematic off-target effects. These results will help guide the proper use and analysis of loss-of-function reagents for the determination of gene function.