Efficient CRISPR/Cas9-Mediated Versatile, Predictable, and Donor-Free Gene Knockout in Human Pluripotent Stem Cells.
Efficient CRISPR/Cas9-Mediated Versatile, Predictable, and Donor-Free Gene Knockout in Human Pluripotent Stem Cells.
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高效 CRISPR/Cas9 介导的多功能、可预测且无供体的人类多能干细胞基因敲除
DOI：
10.1016/j.stemcr.2016.07.021
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发表时间：
2016-09-13
影响因子：
5.9
通讯作者：
Zhang, Xiaoqing
中科院分区：
文献类型：
--
作者：
Liu, Zhongliang;Hui, Yi;Shi, Lei;Chen, Zhenyu;Xu, Xiangjie;Chi, Liankai;Fan, Beibei;Fang, Yujiang;Liu, Yang;Ma, Lin;Wang, Yiran;Xiao, Lei;Zhang, Quanbin;Jin, Guohua;Liu, Ling;Zhang, Xiaoqing
人类多能干细胞（hPSC）的功能丧失研究需要有效的方法来损伤感兴趣的基因。在这里，我们介绍了一种无供体的配对gRNA引导的CRISPR/Cas9敲除策略（配对KO），用于hPSC中的高效和快速基因消融。通过配对KO，我们成功地以高双等位基因靶向效率靶向所有感兴趣的基因。更重要的是，在配对KO过程中，切割的DNA主要通过直接末端连接而没有插入/缺失（精确连接）来修复，从而使损伤产物可预测。配对KO对于多个基因的一步靶向仍然是高效的，并且对于微小RNA的靶向也是高效的，而对于超过8 kb的长非编码RNA，核心启动子区的短片段的切割足以消除下游基因转录。这项工作表明，配对KO策略是用于hPSC中编码基因和非编码基因的功能丧失研究的简单且稳健的系统。Paired-KO代表了hPSC中的有效基因切除方法。在Paired-KO中可以容易地预测切割位点和连接模式。Paired-KO适合于编码和非编码基因的功能丧失研究。启动子去除足以根除下游基因转录。和同事表明，在人多能干细胞中，两个相邻的双链断裂优先通过精确连接而不插入/缺失来修复。因此，这种策略将作为一种有效的方式删除基因片段，并代表了一个精确的方法，在人类多能干细胞的编码和非编码基因的功能丧失的研究。
Loss-of-function studies in human pluripotent stem cells (hPSCs) require efficient methodologies for lesion of genes of interest. Here, we introduce a donor-free paired gRNA-guided CRISPR/Cas9 knockout strategy (paired-KO) for efficient and rapid gene ablation in hPSCs. Through paired-KO, we succeeded in targeting all genes of interest with high biallelic targeting efficiencies. More importantly, during paired-KO, the cleaved DNA was repaired mostly through direct end joining without insertions/deletions (precise ligation), and thus makes the lesion product predictable. The paired-KO remained highly efficient for one-step targeting of multiple genes and was also efficient for targeting of microRNA, while for long non-coding RNA over 8 kb, cleavage of a short fragment of the core promoter region was sufficient to eradicate downstream gene transcription. This work suggests that the paired-KO strategy is a simple and robust system for loss-of-function studies for both coding and non-coding genes in hPSCs. Paired-KO represents an efficient gene ablation methodology in hPSCs The cleavage sites and ligation pattern can be easily predicted in paired-KO Paired-KO fits for loss-of-function studies for both coding and non-coding genes Promoter removal is sufficient to eradicate downstream gene transcription In this article, Zhang, Liu, Jin, and colleagues show that two adjacent double-strand breaks are preferentially repaired through precise ligation without insertions/deletions in human pluripotent stem cells. This strategy will thus serve as an efficient way to delete a gene fragment and represents a precise methodology for loss-of-function studies of both coding and non-coding genes in human pluripotent stem cells.
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影响因子：
48
作者：
Mali, Prashant;Esvelt, Kevin M.;Church, George M.
通讯作者：
Church, George M.
影响因子：
46.9
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23.9
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Xiao A;Wang Z;Hu Y;Wu Y;Luo Z;Yang Z;Zu Y;Li W;Huang P;Tong X;Zhu Z;Lin S;Zhang B
通讯作者：
Zhang B
影响因子：
16.8
作者：
Brogna, Saverio;Wen, Jikai
通讯作者：
Wen, Jikai