iKA-CRISPR hESCs for inducible and multiplex orthogonal gene knockout and activation

FEBS Lett. 2018 Jul;592(13):2238-2247. doi: 10.1002/1873-3468.13127. Epub 2018 Jun 15.

Abstract

Human embryonic stem cells (hESCs) have a wide range of applications in early human embryonic development mimics, disease modeling, and cell therapy. To fulfill these applications, we established hESCs for inducible and multiplex orthogonal gene knockout and activation, which we named iKA-CRISPR hESCs. In cells, when complexed with a short guide RNA containing a 14-bp target sequence (14-bp gRNA) or a long 20-bp gRNA, the doxycycline-induced Cas9-p300 protein could activate gene transcription or cleave genomic DNA, respectively. We also demonstrate using iKA-CRISPR hESCs that knockout of OCT4 promoted differentiation, and developmentally relevant microRNAs and transcription factors could be efficiently activated. Thus, iKA-CRISPR hESCs provide a convenient platform to control gene expression networks and, therefore, facilitate the applications of hESCs in basic and translational biomedical research.

Keywords: CRISPR; hESCs; orthogonal gene knockout and activation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CRISPR-Cas Systems / genetics*
  • Cell Differentiation / genetics
  • Cells, Cultured
  • Gene Expression / genetics
  • Gene Knockout Techniques*
  • Human Embryonic Stem Cells / metabolism*
  • Human Embryonic Stem Cells / physiology
  • Humans
  • Mice
  • Mice, Transgenic
  • RNA, Guide, CRISPR-Cas Systems / genetics
  • RNA, Guide, CRISPR-Cas Systems / metabolism
  • Transcriptional Activation / physiology

Substances

  • RNA, Guide, CRISPR-Cas Systems