biostudies-literaturePandit BNHGRI NIH HHSNIGMS NIH HHS2551-2556https://www.ebi.ac.uk/biostudies/studies/S-EPMC12715751biostudies-literatureUnknown36(12)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 activitBioconjugate chemistryEffects of N&lt;sup&gt;6&lt;/sup&gt;-Methyladenosine (m&lt;sup&gt;6&lt;/sup&gt;A) and 5-Methylcytosine (m&lt;sup&gt;5&lt;/sup&gt;C) Modifications in the Guide Region of CRISPR RNA on Cas12a Nuclease Activity.PMC12715751R35 GM156250R21 HG012257Yigit MVRoyzen MPandit BYang QHanson EDagci HfalseEffects of N&lt;sup&gt;6&lt;/sup&gt;-Methyladenosine (m&lt;sup&gt;6&lt;/sup&gt;A) and 5-Methylcytosine (m&lt;sup&gt;5&lt;/sup&gt;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 activit2025-01-01T00:00:00Z2025 Dec2026-06-06T04:48:54.307Z2026-05-25T03:12:10.991ZS-EPMC127157514134623710.1021/acs.bioconjchem.5c00498