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

ABSTRACT: 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 (T_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 activit

SUBMITTER: Pandit B 

PROVIDER: S-EPMC12715751 | biostudies-literature | 2025 Dec

REPOSITORIES: biostudies-literature