Work

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

Year: 2025

Type: article

Abstract: High Resolution Image Download MS PowerPoint Slide 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, N 6 -methyladenosine (m 6 A) and 5-methylcytosine (m 5 C), introduced into the different positions of the guide region of a crRNA. Melting temperature ( T m ) analysis showed that m 6 A had a minimal impact on RNA–DNA duplex stability. In contrast, the incorporation of m 5 C 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 T m increase with m 5 C, m 6 A incorporation led to a faster fluorescence recovery rate than that with m 5 C.

Source: Bioconjugate Chemistry

DOI: 10.1021/acs.bioconjchem.5c00498

Authors: Bhoomika Pandit, Emmett Hanson, Hilal Dagci, Qingying Yang, Mehmet V. Yigit, Maksim Royzen

Institution: University at Albany, State University of New York

Language: English

Topic: CRISPR and Genetic Engineering

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: hybrid

Estimated APC paid: $4,500

Funders: National Institute of Food and Agriculture, National Human Genome Research Institute, National Institute of General Medical Sciences