Institute of Clinical Biochemistry, Hannover Medical School, Hannover, Germany.
Curr Protoc Stem Cell Biol. 2020 Sep;54(1):e116. doi: 10.1002/cpsc.116.
During the past decade, RNA-guided Cas9 nuclease from microbial clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) has become a powerful tool for gene editing of human pluripotent stem cells (PSCs). Using paired CRISPR/Cas9 nickases (CRISPR/Cas9n) it is furthermore possible to reduce off-target effects that may typically occur with traditional CRISPR/Cas9 systems while maintaining high on-target efficiencies. With this technology and a well-designed homology-directed repair vector (HDR), we are now able to integrate transgenes into specific gene loci of PSCs in an allele conserving way. In this protocol we describe CRISPR/Cas9n design and homology directed repair vector design, transfection of human pluripotent stem cells and selection and expansion of generated cell clones. © 2020 The Authors. Basic Protocol 1: Repair template design and CRISPR/Cas9n construction Basic Protocol 2: Transfection of human pluripotent stem cells by electroporation Basic Protocol 3: Genotyping of generated cell clones.
在过去的十年中，来自微生物成簇规律间隔短回文重复（CRISPR/Cas9）的 RNA 指导 Cas9 核酸酶已成为编辑人类多能干细胞（PSCs）基因的有力工具。使用成对的 CRISPR/Cas9 切口酶（CRISPR/Cas9n），还可以降低传统 CRISPR/Cas9 系统中通常会出现的脱靶效应，同时保持高靶效率。有了这项技术和精心设计的同源定向修复载体（HDR），我们现在能够以保持等位基因的方式将转基因整合到 PSCs 的特定基因座中。在本方案中，我们描述了 CRISPR/Cas9n 的设计和同源定向修复载体的设计、人多能干细胞的转染以及生成细胞克隆的选择和扩增。© 2020 作者。基本方案 1：修复模板设计和 CRISPR/Cas9n 构建 基本方案 2：电穿孔转染人多能干细胞 基本方案 3：生成细胞克隆的基因分型。
