Efficient CRISPR/Cas9-Mediated Versatile, Predictable, and Donor-Free Gene Knockout in Human Pluripotent Stem Cells.
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.
Shanghai Tenth People's Hospital, and Neuroregeneration Key Laboratory of Shanghai Universities, Tongji University School of Medicine, Shanghai 200092, China.
Zhongliang
Z
Liu
Department of Anatomy and Neurobiology, the Jiangsu Key Laboratory of Neuroregeneration, Nantong University, 19 Qixiu Road, Nantong, Jiangsu 226001, China.
Yi
Y
Hui
College of Animal Science and Zhejiang University School of Medicine, Zhejiang University, Hangzhou 310058, China.
Lei
L
Shi
Shanghai Tenth People's Hospital, and Neuroregeneration Key Laboratory of Shanghai Universities, Tongji University School of Medicine, Shanghai 200092, China.
Zhenyu
Z
Chen
Shanghai Tenth People's Hospital, and Neuroregeneration Key Laboratory of Shanghai Universities, Tongji University School of Medicine, Shanghai 200092, China.
Xiangjie
X
Xu
Shanghai Tenth People's Hospital, and Neuroregeneration Key Laboratory of Shanghai Universities, Tongji University School of Medicine, Shanghai 200092, China.
Liankai
L
Chi
Shanghai Tenth People's Hospital, and Neuroregeneration Key Laboratory of Shanghai Universities, Tongji University School of Medicine, Shanghai 200092, China.
Beibei
B
Fan
Shanghai Tenth People's Hospital, and Neuroregeneration Key Laboratory of Shanghai Universities, Tongji University School of Medicine, Shanghai 200092, China.
Yujiang
Y
Fang
Shanghai Tenth People's Hospital, and Neuroregeneration Key Laboratory of Shanghai Universities, Tongji University School of Medicine, Shanghai 200092, China.
Yang
Y
Liu
Shanghai Tenth People's Hospital, and Neuroregeneration Key Laboratory of Shanghai Universities, Tongji University School of Medicine, Shanghai 200092, China.
Lin
L
Ma
Shanghai Tenth People's Hospital, and Neuroregeneration Key Laboratory of Shanghai Universities, Tongji University School of Medicine, Shanghai 200092, China.
Yiran
Y
Wang
College of Animal Science and Zhejiang University School of Medicine, Zhejiang University, Hangzhou 310058, China.
Lei
L
Xiao
Shanghai Tenth People's Hospital, and Neuroregeneration Key Laboratory of Shanghai Universities, Tongji University School of Medicine, Shanghai 200092, China.
Quanbin
Q
Zhang
Department of Anatomy and Neurobiology, the Jiangsu Key Laboratory of Neuroregeneration, Nantong University, 19 Qixiu Road, Nantong, Jiangsu 226001, China. Electronic address: jguohua@ntu.edu.cn.
Guohua
G
Jin
Shanghai Tenth People's Hospital, and Neuroregeneration Key Laboratory of Shanghai Universities, Tongji University School of Medicine, Shanghai 200092, China; Tongji University Advanced Institute of Translational Medicine, Tongji University School of Medicine, 1239 Siping Road, Shanghai 200092, China. Electronic address: lliu@tongji.edu.cn.
Ling
L
Liu
Shanghai Tenth People's Hospital, and Neuroregeneration Key Laboratory of Shanghai Universities, Tongji University School of Medicine, Shanghai 200092, China; Tongji University Advanced Institute of Translational Medicine, Tongji University School of Medicine, 1239 Siping Road, Shanghai 200092, China; The Collaborative Innovation Center for Brain Science, Tongji University, Shanghai 200092, China. Electronic address: xqzhang@tongji.edu.cn.
Xiaoqing
X
Zhang
D001483:Base Sequence; D064113:CRISPR-Cas Systems; D002454:Cell Differentiation; D002460:Cell Line; D053595:Embryonic Stem Cells; D055786:Gene Knockout Techniques; D018390:Gene Targeting; D006801:Humans; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D000094704:RNA, Guide, CRISPR-Cas Systems; D062085:RNA, Long Noncoding / Q000235:genetics; D051900:Smad3 Protein / Q000172:deficiency / Q000235:genetics; D051176:beta Catenin / Q000172:deficiency / Q000235:genetics
C495270:CTNNB1 protein, human; D000094704:RNA, Guide, CRISPR-Cas Systems; D062085:RNA, Long Noncoding; D051900:Smad3 Protein; D051176:beta Catenin
CRISPR/Cas9; gene editing; human pluripotent stem cells; non-coding RNA