Scientific Reports (May 2017)

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
https://doi.org/10.1038/s41598-017-02456-y
Journal volume & issue
Vol. 7, no. 1
pp. 1 – 11

Abstract

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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.