CRISPR deactivation in mammalian cells using photocleavable guide RNAs

STAR Protoc. 2021 Oct 20;2(4):100909. doi: 10.1016/j.xpro.2021.100909. eCollection 2021 Dec 17.

Abstract

The ability to deactivate CRISPR-Cas systems on demand would improve the safety and applicability of genome editing. Here, we detail a protocol using photocleavable guide RNAs (pcRNAs) to deactivate CRISPR-Cas9 inside cells. We verify that deactivation is both rapid and complete by checking for insertion-deletion (indel) mutations using Sanger sequencing. This protocol will be useful for researchers interested in using pcRNAs to improve genome editing specificity, characterize the timescales of genome editing, and study cellular DNA damage responses. For complete details on the use and execution of this protocol, please refer to Zou et al. (2021).

Keywords: Biotechnology and bioengineering; CRISPR; Cell Biology; Cell-based Assays; Genomics; Molecular Biology; Molecular/Chemical Probes; Sequencing.

Publication types

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

MeSH terms

  • CRISPR-Associated Protein 9 / genetics
  • CRISPR-Associated Protein 9 / metabolism
  • CRISPR-Associated Protein 9 / radiation effects
  • CRISPR-Cas Systems / genetics*
  • Gene Editing / methods*
  • HEK293 Cells
  • Humans
  • INDEL Mutation / genetics
  • Photolysis
  • RNA, Guide, CRISPR-Cas Systems* / genetics
  • RNA, Guide, CRISPR-Cas Systems* / metabolism
  • RNA, Guide, CRISPR-Cas Systems* / radiation effects

Substances

  • RNA, Guide, CRISPR-Cas Systems
  • CRISPR-Associated Protein 9