Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation.
Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation.
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DOI：
10.3791/53583
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发表时间：
2016-02-02
期刊：
影响因子：
--
通讯作者：
Hockemeyer DF
中科院分区：
文献类型：
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作者：
Blair JD;Bateup HS;Hockemeyer DF
人类多能干细胞(HPSCs)的基因组编辑为了解人类发育和研究疾病的病理生理学提供了一个受基因控制和临床相关的平台。通过使用位点特异性核酸酶(SSN)进行基因组编辑，可以快速获得在其他等基因环境中具有特定遗传变化的新的hPSC株系。锌指核酸酶(ZFN)、转录激活物样效应核酸酶(TALEN)和簇状规则间隔短回文重复序列(CRISPR)/Cas9是最常用的SSN。所有这些核酸酶的功能都是通过在特定位置引入双链DNA断裂来发挥作用，从而促进基因组位置的精确基因编辑。SSN冥想的基因组编辑利用细胞的两种内源性DNA修复机制--非同源末端连接(NHEJ)和同源定向修复(HDR)--引入插入/缺失突变或在双链断裂部位使用同源修复模板改变基因组。HPSCs的电穿孔是一种有效的方法，可以将SSN和含有转基因的修复模板(如荧光报告基因和抗生素耐药盒)转化为SSN。在电穿孔后，通过选择抗生素耐药性，可以仅分离那些包含修复构建体的hPSCs。机械分离hPSC克隆，并通过基因分型确认目标位置的正确整合，可以分离出正确靶向和基因同质的细胞系。在这里，通过使用所有三个SSN平台将EGFP和嘌呤霉素抗性构建物整合到人多能干细胞的AAVS1安全港基因座中，证明了该协议的有效性。
Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human development and investigate the pathophysiology of disease. By employing site-specific nucleases (SSNs) for genome editing, the rapid derivation of new hPSC lines harboring specific genetic alterations in an otherwise isogenic setting becomes possible. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 are the most commonly used SSNs. All of these nucleases function by introducing a double stranded DNA break at a specified site, thereby promoting precise gene editing at a genomic locus. SSN-meditated genome editing exploits two of the cell's endogenous DNA repair mechanisms, non-homologous end joining (NHEJ) and homology directed repair (HDR), to either introduce insertion/deletion mutations or alter the genome using a homologous repair template at the site of the double stranded break. Electroporation of hPSCs is an efficient means of transfecting SSNs and repair templates that incorporate transgenes such as fluorescent reporters and antibiotic resistance cassettes. After electroporation, it is possible to isolate only those hPSCs that incorporated the repair construct by selecting for antibiotic resistance. Mechanically separating hPSC colonies and confirming proper integration at the target site through genotyping allows for the isolation of correctly targeted and genetically homogeneous cell lines. The validity of this protocol is demonstrated here by using all three SSN platforms to incorporate EGFP and a puromycin resistance construct into the AAVS1 safe harbor locus in human pluripotent stem cells.