Reversible RNA Acylation Using Bio-Orthogonal Chemistry Enables Temporal Control of CRISPR-Cas9 Nuclease Activity.

ABSTRACT: The CRISPR-Cas9 system is a widely popular tool for genome engineering. There is strong interest in developing tools for temporal control of CRISPR-Cas9 activity to address some of the challenges and to broaden the scope of potential applications. In this work, we describe a bio-orthogonal chemistry-based approach to control nuclease activity with temporal precision. We report a trans-cyclooctene (TCO)-acylimidazole reagent that acylates 2'-OH groups of RNA. Poly acylation ("cloaking") of RNA was optimized in vitro using a model 18-nt oligonucleotide, as well as CRISPR single guide RNA (sgRNA). Two hours of treatment completely inactivated sgRNA for Cas9-assisted DNA cleavage. Nuclease activity was restored upon addition of tetrazine, which removes the TCO moieties via a two-

SUBMITTER: Pandit B 

PROVIDER: S-EPMC11334111 | biostudies-literature | 2024 Aug

REPOSITORIES: biostudies-literature