Cas9 deactivation with photocleavable guide RNAs

Roger S Zou; Yang Liu; Bin Wu; Taekjip Ha

doi:10.1016/j.molcel.2021.02.007

Cas9 deactivation with photocleavable guide RNAs

Mol Cell. 2021 Apr 1;81(7):1553-1565.e8. doi: 10.1016/j.molcel.2021.02.007. Epub 2021 Mar 3.

Authors

Roger S Zou¹, Yang Liu², Bin Wu², Taekjip Ha³

Affiliations

¹ Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21205, USA.
² Department of Biophysics and Biophysical Chemistry, Johns Hopkins University, Baltimore, MD 21205, USA.
³ Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21205, USA; Department of Biophysics and Biophysical Chemistry, Johns Hopkins University, Baltimore, MD 21205, USA; Department of Biophysics, Johns Hopkins University, Baltimore, MD 21205, USA; Howard Hughes Medical Institute, Baltimore, MD 21205, USA. Electronic address: tjha@jhu.edu.

Abstract

Precise control of CRISPR-Cas9 would improve its safety and applicability. Controlled CRISPR inhibition is a promising approach but is complicated by separate inhibitor delivery, incomplete deactivation, and slow kinetics. To overcome these obstacles, we engineered photocleavable guide RNAs (pcRNAs) that endow Cas9 nucleases and base editors with a built-in mechanism for light-based deactivation. pcRNA enabled the fastest (<1 min) and most complete (<1% residual indels) approach for Cas9 deactivation. It also exhibited significantly enhanced specificity with wild-type Cas9. Time-resolved deactivation revealed that 12-36 h of Cas9 activity or 2-4 h of base editor activity was sufficient to achieve high editing efficiency. pcRNA is useful for studies of the cellular response to DNA damage by abolishing sustained cycles of damage and repair that would otherwise desynchronize response trajectories. Together, pcRNA expands the CRISPR toolbox for precision genome editing and studies of DNA damage and repair.

Keywords: CRISPR; Cas9; DNA repair; deactivation; genome editing; specificity.

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 / chemistry*
CRISPR-Associated Protein 9 / metabolism
CRISPR-Cas Systems*
DNA Damage*
Gene Editing*
RNA, Guide, CRISPR-Cas Systems / chemistry*
RNA, Guide, CRISPR-Cas Systems / metabolism

Substances

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

Abstract

Publication types

MeSH terms

Substances

Grants and funding