1] State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing 100871, China [2].
State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing 100871, China.
Nature. 2014 May 22;509(7501):487-91. doi: 10.1038/nature13166. Epub 2014 Apr 9.
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.
靶向基因组编辑技术是研究生物学和疾病的强大工具，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比，基于完全丧失基因表达的大规模筛选方法现在才刚刚开始开发。在这里，我们报告了在人类细胞中开发基于聚焦 CRISPR/Cas（成簇的、规律间隔的短回文重复序列/CRISPR 相关）慢病毒文库的方法，以及基于功能筛选和高通量测序分析的基因鉴定方法。使用敲除文库筛选，我们成功鉴定了炭疽和白喉毒素使细胞中毒所必需的宿主基因，这些基因通过功能验证得到了确认。这种强大的遗传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，还将能够发现参与其他生物过程的基因。
