CRISPR/Cas9-Mediated Mutagenesis of Human Pluripotent Stem Cells in Defined Xeno-Free E8 Medium.
CRISPR/Cas9-Mediated Mutagenesis of Human Pluripotent Stem Cells in Defined Xeno-Free E8 Medium.
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在确定的无异源 E8 培养基中 CRISPR/Cas9 介导的人类多能干细胞诱变。
DOI：
10.1007/978-1-4939-6472-7_5
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
Huangfu,Danwei
中科院分区：
文献类型：
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作者：
Soh,Chew-Li;Huangfu,Danwei
最近出现的工程核酸酶，包括CRISPR/Cas9系统，极大地促进了人类多能干细胞(HPSCs)的基因组操作。除了促进基于hPSC的疾病研究外，基因组工程在hPSC中的应用也为细胞替代治疗开辟了新的途径。为了提高基于hPSC的研究的一致性和重复性，并满足临床翻译的安全性和法规要求，有必要使用明确的、无异种的细胞培养系统。本章描述了CRISPR/Cas9基因组编辑的协议，该系统基于可诱导的Cas9 hPSC系统，使用在化学定义的、无异种的E8培养基中培养的细胞，在重组人Vitronectin底物上进行培养。我们详细介绍了CRISPR引导RNA的设计和导入、菌落选择以及克隆突变系的扩展和验证的程序，所有这些都在这种完全定义的培养条件下进行。这些方法可以应用于hPSC的广泛基因组工程应用，包括那些利用不同类型的位点特异性核酸酶的应用，如锌指核酸酶(ZFN)和TALEN，并形成朝着这些细胞的临床实用更近的一步。
The recent advent of engineered nucleases including the CRISPR/Cas9 system has greatly facilitated genome manipulation in human pluripotent stem cells (hPSCs). In addition to facilitating hPSC-based disease studies, the application of genome engineering in hPSCs has also opened up new avenues for cell replacement therapy. To improve consistency and reproducibility of hPSC-based studies, and to meet the safety and regulatory requirements for clinical translation, it is necessary to use a defined, xeno-free cell culture system. This chapter describes protocols for CRISPR/Cas9 genome editing in an inducible Cas9 hPSC-based system, using cells cultured in chemically defined, xeno-free E8 Medium on a recombinant human vitronectin substrate. We detail procedures for the design and transfection of CRISPR guide RNAs, colony selection, and the expansion and validation of clonal mutant lines, all within this fully defined culture condition. These methods may be applied to a wide range of genome-engineering applications in hPSCs, including those that utilize different types of site-specific nucleases such as zinc finger nucleases (ZFNs) and TALENs, and form a closer step towards clinical utility of these cells.