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.

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*