Genome engineering using the CRISPR-Cas9 system
Genome engineering using the CRISPR-Cas9 system
复制标题
DOI：
10.1038/nprot.2013.143
复制
发表时间：
2013-11-01
期刊：
影响因子：
14.8
通讯作者：
Zhang, Feng
中科院分区：
文献类型：
--
作者：
Ran, F. Ann;Hsu, Patrick D.;Zhang, Feng
靶向核酸酶是高精度介导基因组改变的有力工具。来自微生物成簇规则间隔短回文重复序列（CRISPR）适应性免疫系统的RNA引导的Cas9核酸酶可用于通过简单地在其引导RNA内指定20-nt靶向序列来促进真核细胞中的有效基因组工程化。在这里，我们描述了一组用于在哺乳动物细胞中通过非同源末端连接（NHEJ）或同源定向修复（HDR）进行Cas9介导的基因组编辑的工具，以及用于下游功能研究的修饰细胞系的生成。为了使脱靶切割最小化，我们进一步描述了使用Cas9切口酶突变体与配对的指导RNA的双切口策略。该方案为靶位点的选择、切割效率的评价和脱靶活性的分析提供了实验来源的指导。从目标设计开始，基因修饰可以在短短1-2周内实现，并且修饰的克隆细胞系可以在2-3周内衍生。
Targeted nucleases are powerful tools for mediating genome alteration with high precision. The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA. Here we describe a set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation of modified cell lines for downstream functional studies. To minimize off-target cleavage, we further describe a double-nicking strategy using the Cas9 nickase mutant with paired guide RNAs. This protocol provides experimentally derived guidelines for the selection of target sites, evaluation of cleavage efficiency and analysis of off-target activity. Beginning with target design, gene modifications can be achieved within as little as 1-2 weeks, and modified clonal cell lines can be derived within 2-3 weeks.