Effects of N6-Methyladenosine (m6A) and 5-Methylcytosine (m5C) Modifications in the Guide Region of CRISPR RNA on Cas12a Nuclease Activity.

CRISPR-Cas12a is a versatile biosensing platform that detects sequence-specific DNA or RNA targets via a CRISPR RNA (crRNA) guide. While Cas12a's specificity is dictated by its crRNA, chemical modifications within the crRNA can influence nuclease performance. Here, we examined the effects of two well-known RNA modifications, N6-methyladenosine (m6A) and 5-methylcytosine (m5C), introduced into the different positions of the guide region of a crRNA. Melting temperature (Tm) analysis showed that m6A had a minimal impact on RNA-DNA duplex stability. In contrast, the incorporation of m5C residues stabilized the duplex. Using a fluorescence recovery assay, we found that both modifications preserved Cas12a's nuclease activity, indicating that small thermodynamic shifts in duplex formation are insufficient to disrupt its catalytic function. Despite the greater Tm increase with m5C, m6A incorporation led to a faster fluorescence recovery rate than that with m5C.