Effects of N ⁶ -Methyladenosine (m ⁶ A) and 5-Methylcytosine (m ⁵ C) 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 (<i>T</i>_m) 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 <i>T</i>_m increase with m⁵C, m⁶A incorporation led to a faster fluorescence recovery rate than that with m⁵C.