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
Shanghai Tenth People's Hospital, and Neuroregeneration Key Laboratory of Shanghai Universities, Tongji University School of Medicine, Shanghai 200092, China.
Department of Anatomy and Neurobiology, the Jiangsu Key Laboratory of Neuroregeneration, Nantong University, 19 Qixiu Road, Nantong, Jiangsu 226001, China.
Stem Cell Reports. 2016 Sep 13;7(3):496-507. doi: 10.1016/j.stemcr.2016.07.021. Epub 2016 Sep 1.
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.
对人类多能干细胞（hPSC）进行功能丧失研究需要高效的方法来损伤感兴趣的基因。在此，我们介绍一种无供体的双gRNA引导的CRISPR/Cas9敲除策略（配对敲除），用于在hPSC中高效快速地进行基因敲除。通过配对敲除，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，在配对敲除过程中，切割后的DNA大多通过直接末端连接进行修复，无插入/缺失（精确连接），因此使损伤产物具有可预测性。配对敲除对于多基因的一步靶向仍然高效，对微小RNA的靶向也有效，而对于超过8 kb的长链非编码RNA，核心启动子区域的短片段切割就足以消除下游基因转录。这项工作表明，配对敲除策略是用于hPSC中编码和非编码基因功能丧失研究的简单且强大的系统。
