CRISPR-Switch regulates sgRNA activity by Cre recombination for sequential editing of two loci

Nat Commun. 2019 Nov 29;10(1):5454. doi: 10.1038/s41467-019-13403-y.

Abstract

CRISPR-Cas9 is an efficient and versatile tool for genome engineering in many species. However, inducible CRISPR-Cas9 editing systems that regulate Cas9 activity or sgRNA expression often suffer from significant limitations, including reduced editing capacity, off-target effects, or leaky expression. Here, we develop a precisely controlled sgRNA expression cassette that can be combined with widely-used Cre systems, termed CRISPR-Switch (SgRNA With Induction/Termination by Cre Homologous recombination). Switch-ON facilitates controlled, rapid induction of sgRNA activity. In turn, Switch-OFF-mediated termination of editing improves generation of heterozygous genotypes and can limit off-target effects. Furthermore, we design sequential CRISPR-Switch-based editing of two loci in a strictly programmable manner and determined the order of mutagenic events that leads to development of glioblastoma in mice. Thus, CRISPR-Switch substantially increases the versatility of gene editing through precise and rapid switching ON or OFF sgRNA activity, as well as switching OVER to secondary sgRNAs.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CRISPR-Cas Systems
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Gene Editing / methods*
  • Genetic Engineering
  • Homologous Recombination
  • Integrases
  • Mice
  • Mouse Embryonic Stem Cells / metabolism*
  • Mutagenesis
  • RNA Polymerase III
  • RNA, Guide, CRISPR-Cas Systems / genetics*

Substances

  • RNA, Guide, CRISPR-Cas Systems
  • Cre recombinase
  • Integrases
  • RNA Polymerase III