Reversible Disruption of Specific Transcription Factor-DNA Interactions Using CRISPR/Cas9

Mol Cell. 2019 May 2;74(3):622-633.e4. doi: 10.1016/j.molcel.2019.04.011.

Abstract

The control of gene expression by transcription factor binding sites frequently determines phenotype. However, it is difficult to determine the function of single transcription factor binding sites within larger transcription networks. Here, we use deactivated Cas9 (dCas9) to disrupt binding to specific sites, a method we term CRISPRd. Since CRISPR guide RNAs are longer than transcription factor binding sites, flanking sequence can be used to target specific sites. Targeting dCas9 to an Oct4 site in the Nanog promoter displaced Oct4 from this site, reduced Nanog expression, and slowed division. In contrast, disrupting the Oct4 binding site adjacent to Pax6 upregulated Pax6 transcription and disrupting Nanog binding its own promoter upregulated its transcription. Thus, we can easily distinguish between activating and repressing binding sites and examine autoregulation. Finally, multiple guide RNA expression allows simultaneous inhibition of multiple binding sites, and conditionally destabilized dCas9 allows rapid reversibility.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Binding Sites / genetics
  • CRISPR-Associated Protein 9 / genetics
  • CRISPR-Cas Systems / genetics*
  • Gene Expression Regulation / genetics
  • Gene Regulatory Networks
  • Humans
  • Mice
  • Mouse Embryonic Stem Cells / cytology
  • Mouse Embryonic Stem Cells / metabolism
  • Nanog Homeobox Protein / genetics*
  • Octamer Transcription Factor-3 / genetics*
  • PAX6 Transcription Factor / genetics*
  • Promoter Regions, Genetic
  • RNA, Guide / genetics
  • Transcription Factors / genetics
  • Transcriptional Activation / genetics

Substances

  • Nanog Homeobox Protein
  • Nanog protein, mouse
  • Octamer Transcription Factor-3
  • PAX6 Transcription Factor
  • Pax6 protein, mouse
  • Pou5f1 protein, mouse
  • RNA, Guide
  • Transcription Factors
  • CRISPR-Associated Protein 9