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

-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.