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

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

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

80 Citations (Scopus)

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.

Original language	English
Article number	2320
Journal	Scientific Reports
Volume	7
Issue number	1
DOIs	https://doi.org/10.1038/s41598-017-02456-y
Publication status	Published - 1 Dec 2017
Externally published	Yes

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

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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AU - Xie, Yifang

AU - Wang, Daqi

AU - Lan, Feng

AU - Wei, Gang

AU - Ni, Ting

AU - Chai, Renjie

AU - Liu, Dong

AU - Hu, Shijun

AU - Li, Mingqing

AU - Li, Dajin

AU - Wang, Hongyan

AU - Wang, Yongming

PY - 2017/12/1

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N2 - 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.

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

Abstract

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