Issue 22, 2023

Regulation of the CRISPR-Cas12a system by methylation and demethylation of guide RNA

Abstract

Chemical modifications of CRISPR-Cas nucleases help decrease off-target editing and expand the biomedical applications of CRISPR-based gene manipulation tools. Here, we found that epigenetic modifications of guide RNA, such as m6A and m1A methylation, can effectively inhibit both the cis- and trans-DNA cleavage activities of CRISPR-Cas12a. The underlying mechanism is that methylations destabilize the secondary and tertiary structure of gRNA which prevents the assembly of the Cas12a-gRNA nuclease complex, leading to decreased DNA targeting ability. A minimum of three adenine methylated nucleotides are required to completely inhibit the nuclease activity. We also demonstrate that these effects are reversible through the demethylation of gRNA by demethylases. This strategy has been used in the regulation of gene expression, demethylase imaging in living cells and controllable gene editing. The results demonstrate that the methylation-deactivated and demethylase-activated strategy is a promising tool for regulation of the CRISPR-Cas12a system.

Graphical abstract: Regulation of the CRISPR-Cas12a system by methylation and demethylation of guide RNA

Supplementary files

Article information

Article type
Edge Article
Submitted
06 Feb 2023
Accepted
08 May 2023
First published
09 May 2023
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY-NC license

Chem. Sci., 2023,14, 5945-5955

Regulation of the CRISPR-Cas12a system by methylation and demethylation of guide RNA

Z. Hu, A. Sun, J. Yang, G. Naz, G. Sun, Z. Li, J. Gogo Liu, S. Zhang and X. Zhang, Chem. Sci., 2023, 14, 5945 DOI: 10.1039/D3SC00629H

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