Yang Luhan, Mali Prashant, Kim-Kiselak Caroline, Church George
Department of Genetics, Harvard Medical School, Boston, MA, USA.
Methods Mol Biol. 2014;1114:245-67. doi: 10.1007/978-1-62703-761-7_16.
The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems have evolved as an adaptive surveillance and defense mechanism in bacteria and archaea that uses short RNAs to direct degradation of foreign genetic elements. Here, we present our protocol for utilizing the S. pyogenes type II bacterial CRISPR system to achieve sequence-specific genome alterations in human cells. In principle, any genomic sequence of the form N₁₉NGG can be targeted with the generation of custom guide RNA (gRNA) which functions to direct the Cas9 protein to genomic targets and induce DNA cleavage. Here, we describe our methods for designing and generating gRNA expression constructs either singly or in a multiplexed manner, as well as optimized protocols for the delivery of Cas9-gRNA components into human cells. Genomic alterations at the target site are then introduced either through nonhomologous end joining (NHEJ) or through homologous recombination (HR) in the presence of an appropriate donor sequence. This RNA-guided editing tool offers greater ease of customization and synthesis in comparison to existing sequence-specific endonucleases and promises to become a highly versatile and multiplexable human genome engineering platform.
成簇规律间隔短回文重复序列（CRISPR）及其相关（Cas）系统已演化为细菌和古细菌中的一种适应性监测和防御机制，该机制利用短RNA引导对外源遗传元件的降解。在此，我们展示了利用化脓性链球菌II型细菌CRISPR系统在人类细胞中实现序列特异性基因组改变的方案。原则上，任何形式为N₁₉NGG的基因组序列都可以通过生成定制的向导RNA（gRNA）来靶向，该gRNA的作用是引导Cas9蛋白靶向基因组靶点并诱导DNA切割。在此，我们描述了单独或以多重方式设计和生成gRNA表达构建体的方法，以及将Cas9-gRNA组件导入人类细胞的优化方案。然后，通过非同源末端连接（NHEJ）或在存在合适供体序列的情况下通过同源重组（HR）在靶位点引入基因组改变。与现有的序列特异性核酸内切酶相比，这种RNA引导的编辑工具具有更高的定制性和合成便利性，有望成为一个高度通用且可多重化的人类基因组工程平台。
