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

Yifang Xie; Daqi Wang; Feng Lan; Gang Wei; Ting Ni; Renjie Chai; Dong Liu; Shijun Hu; Mingqing Li; Dajin Li; Hongyan Wang; Yongming Wang

doi:10.1038/s41598-017-02456-y

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

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

Authors

Yifang Xie¹, Daqi Wang², Feng Lan³, Gang Wei², Ting Ni², Renjie Chai⁴, Dong Liu⁵, Shijun Hu⁶, Mingqing Li⁷, Dajin Li⁷, Hongyan Wang^{8

9}, Yongming Wang^{10

11}

Affiliations

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

CRISPR-Cas Systems
Cell Differentiation / genetics*
Gene Editing*
Gene Knockout Techniques / methods
Genetic Vectors / genetics
Humans
INDEL Mutation / genetics
Plasmids / genetics*
Pluripotent Stem Cells / cytology
Pluripotent Stem Cells / metabolism*