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

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-step process ("uncloaking"). The approach was applied to target the GFP gene in live HEK293 cells. GFP expression was analyzed by flow cytometry. In the future, we anticipate that our approach will be useful in the field of developmental biology, by enabling investigation of genes of interest at different stages of an organism's development.