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Sci Rep. 2017 May 24;7(1):2320. doi: 10.1038/s41598-017-02456-y.

An episomal vector-based CRISPR/Cas9 system for highly efficient gene knockout in human pluripotent stem cells.

Xie Y1, Wang D2, Lan F3, Wei G2, Ni T2, Chai R4, Liu D5, Hu S6, Li M7, Li D7, Wang H8,9, Wang Y10,11.

Author information

1
Institute of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, 200032, China.
2
The State Key Laboratory of Genetic Engineering and MOE Key Laboratory of Contemporary Anthropology, School of Life Sciences, Fudan University, Shanghai, 200438, China.
3
Beijing Anzhen Hospital, Beijing Insitute of Heart Lung and Blood Vessel Disease, Capital Medical University, Beijing, 100029, China.
4
Co-innovation Center of Neuro regeneration, Key Laboratory for Developmental Genes and Human Disease, Ministry of Education, Institute of Life Sciences, Southeast University, Nanjing, 210096, China.
5
Co-innovation Center of Neuroregeneration, Jiangsu Key Laboratory of Neuroregeneration, Nantong University, Nantong, 226001, China.
6
Institute for Cardiovascular Science & Department of Cardiovascular Surgery of the First Affiliated Hospital, Soochow University, Soochow, 215007, China.
7
The Key Lab of Reproduction Regulation of NPFPC in SIPPR, Institute of Reproduction & Development in Obstetrics & Gynecology Hospital, Fudan University, Shanghai, 200011, China.
8
The Key Lab of Reproduction Regulation of NPFPC in SIPPR, Institute of Reproduction & Development in Obstetrics & Gynecology Hospital, Fudan University, Shanghai, 200011, China. wanghy@fudan.edu.cn.
9
Children's Hospital of Fudan University, 399 Wanyuan Road, Shanghai, 201102, China. wanghy@fudan.edu.cn.
10
The State Key Laboratory of Genetic Engineering and MOE Key Laboratory of Contemporary Anthropology, School of Life Sciences, Fudan University, Shanghai, 200438, China. ymw@fudan.edu.cn.
11
The Key Lab of Reproduction Regulation of NPFPC in SIPPR, Institute of Reproduction & Development in Obstetrics & Gynecology Hospital, Fudan University, Shanghai, 200011, China. ymw@fudan.edu.cn.

Abstract

Human pluripotent stem cells (hPSCs) represent a unique opportunity for understanding the molecular mechanisms underlying complex traits and diseases. CRISPR/Cas9 is a powerful tool to introduce genetic mutations into the hPSCs for loss-of-function studies. Here, we developed an episomal vector-based CRISPR/Cas9 system, which we called epiCRISPR, for highly efficient gene knockout in hPSCs. The epiCRISPR system enables generation of up to 100% Insertion/Deletion (indel) rates. In addition, the epiCRISPR system enables efficient double-gene knockout and genomic deletion. To minimize off-target cleavage, we combined the episomal vector technology with double-nicking strategy and recent developed high fidelity Cas9. Thus the epiCRISPR system offers a highly efficient platform for genetic analysis in hPSCs.

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