通过CRISPR/ cas9n介导的同源定向修复设计和衍生多报告多能干细胞系

Q2 Biochemistry, Genetics and Molecular Biology Current Protocols in Stem Cell Biology Pub Date : 2020-07-06 DOI:10.1002/cpsc.116

Rabea Dettmer, Ortwin Naujok

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引用次数: 3

摘要

在过去的十年中，来自微生物聚集规律间隔短回文重复序列(CRISPR/Cas9)的rna引导Cas9核酸酶已成为人类多能干细胞(PSCs)基因编辑的有力工具。使用配对的CRISPR/Cas9缺口酶(CRISPR/Cas9n)，进一步有可能减少传统CRISPR/Cas9系统通常发生的脱靶效应，同时保持高靶向效率。利用这项技术和精心设计的同源定向修复载体(HDR)，我们现在能够以等位基因保护的方式将转基因整合到PSCs的特定基因位点上。在本协议中，我们描述了CRISPR/Cas9n设计和同源定向修复载体设计，转染人类多能干细胞以及选择和扩增生成的细胞克隆。©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

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Design and Derivation of Multi-Reporter Pluripotent Stem Cell Lines via CRISPR/Cas9n-Mediated Homology-Directed Repair

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

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Current Protocols in Stem Cell Biology Biochemistry, Genetics and Molecular Biology-Cell Biology

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期刊介绍： Published in affiliation with the International Society for Stem Cell Research (ISSCR), Current Protocols in Stem Cell Biology (CPSC) covers the most fundamental protocols and methods in the rapidly growing field of stem cell biology. Updated monthly, CPSC will constantly evolve with thelatest developments and breakthroughs in the field. Drawing on the expertise of leading researchers from around the world, Current Protocols in Stem Cell Biology includes methods and insights that will enhance the progress of global research.