High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells
High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells
复制标题
人类细胞功能基因组学 CRISPR/Cas9 文库的高通量筛选
DOI：
10.1038/nature13166
复制
发表时间：
2014-05-22
期刊：
影响因子：
64.8
通讯作者：
Wei, Wensheng
中科院分区：
文献类型：
--
作者：
Zhou, Yuexin;Zhu, Shiyou;Wei, Wensheng
靶向基因组编辑技术是研究生物学和疾病的有力工具，具有广泛的研究应用、、、、、、。与操纵单个基因的工具包的快速发展相反，基于基因表达完全丧失的大规模筛选方法现在才刚刚开始开发。在这里，我们报告了人类细胞中基于 CRISPR/Cas（成簇规则间隔短回文重复/CRISPR 相关）慢病毒文库的开发，以及基于功能筛选和高通量测序分析的基因鉴定方法。通过敲除文库筛选，我们成功鉴定了炭疽和白喉毒素细胞中毒所必需的宿主基因，并通过功能验证得到证实。这种强大的基因筛选策略的广泛应用不仅将有助于快速鉴定对细菌毒性重要的基因，而且还将能够发现参与其他生物过程的基因。
Targeted genome editing technologies are powerful tools for studying biology and disease, and have a broad range of research applications,,,,,,. In contrast to the rapid development of toolkits to manipulate individual genes, large-scale screening methods based on the complete loss of gene expression are only now beginning to be developed,. Here we report the development of a focused CRISPR/Cas-based (clustered regularly interspaced short palindromic repeats/CRISPR-associated) lentiviral library in human cells and a method of gene identification based on functional screening and high-throughput sequencing analysis. Using knockout library screens, we successfully identified the host genes essential for the intoxication of cells by anthrax and diphtheria toxins, which were confirmed by functional validation. The broad application of this powerful genetic screening strategy will not only facilitate the rapid identification of genes important for bacterial toxicity but will also enable the discovery of genes that participate in other biological processes.