Generation and validation of PAX7 reporter lines from human iPS cells using CRISPR/Cas9 technology
Generation and validation of PAX7 reporter lines from human iPS cells using CRISPR/Cas9 technology
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DOI：
10.1016/j.scr.2016.01.003
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发表时间：
2016-03-01
影响因子：
1.2
通讯作者：
Darabi, Radbod
中科院分区：
文献类型：
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作者：
Wu, Jianbo;Hunt, Samuel D.;Darabi, Radbod
诱导多能干细胞向多种组织祖细胞定向分化是近年来研究的热点。因此，组织特异性报告性iPS细胞系的产生可以更好地了解iPS细胞的发育阶段。本技术报告描述了一种利用cas9 -缺口酶系统在人类iPS细胞中产生和验证敲入报告系的有效策略。在这里，我们已经产生了一个敲入人类iPS细胞系的早期肌生成谱系规范基因PAX7。利用CRISPR/Cas9缺失酶对在PAX7基因组位点引入位点特异性双链断裂（DSB），在PAX7基因最后一个外显子的停止密码子前加入一个带有选择标记的2A-GFP报告基因。经过阳性和阴性选择，分离出单细胞衍生的人类iPS克隆，并对其进行测序，以确定报告基因结构的帧内定位。最后，利用核酸酶死亡Cas9激活子（dCas9-VP160）系统，针对PAX7的启动子区域进行瞬时基因诱导，验证GFP报告基因的活性。流式细胞术分析和PAX7和GFP的免疫染色证实了这一点。本技术报告为利用CRISPR/Cas9系统生成和验证敲入报告基因提供了实用指南。Elsevier B.V.出版
Directed differentiation of iPS cells toward various tissue progenitors has been the focus of recent research. Therefore, generation of tissue-specific reporter iPS cell lines provides better understanding of developmental stages in iPS cells. This technical report describes an efficient strategy for generation and validation of knock-in reporter lines in human iPS cells using the Cas9-nickase system.Here, we have generated a knock-in human iPS cell line for the early myogenic lineage specification gene of PAX7. By introduction of site-specific double-stranded breaks (DSB) in the genomic locus of PAX7 using CRISPR/Cas9 nickase pairs, a 2A-GFP reporter with selection markers has been incorporated before the stop codon of the PAX7 gene at the last exon. After positive and negative selection, single cell-derived human iPS clones have been isolated and sequenced for in-frame positioning of the reporter construct.Finally, by using a nuclease-dead Cas9 activator (dCas9-VP160) system, the promoter region of PAX7 has been targeted for transient gene induction to validate the GFP reporter activity. This was confirmed by flow cytometry analysis and immunostaining for PAX7 and GFP. This technical report provides a practical guideline for generation and validation of knock-in reporters using CRISPR/Cas9 system. Published by Elsevier B.V.