"Enhancing CRISPR-Cas12a Activity through Guide RNA Methylation" by Hilal Dagci

Date of Award

Spring 2025

Embargo Period

5-1-2025

Document Type

Master's Thesis

Degree Name

Master of Science (MS)

College/School/Department

Department of Chemistry

Program

Chemistry

First Advisor

Maksim Royzen

Keywords

CRISPR-Cas12a, genome editing, chemically modified guide rna

Subject Categories

Chemistry

Abstract

CRISPR-Cas12a is a rapidly evolving technology for improved gene editing in cells, as well as in vivo. The system, consisting of Cas12a nuclease and approximately 40-nt long guide RNA (gRNA), can efficiently recognize and cut genomic double-stranded DNA (dsDNA). Chemical modifications to the gRNA can potentially improve its dsDNA binding and enhance Cas12a-mediated nuclease activity. In this work, 5-methyl cytidine (m5C) and N6-methyl adenosine (m6A) were explored to improve the binding between gRNA and target DNA. Phosphoramidites of m5C and m6A were used to construct a library of gRNAs using solid phase RNA synthesis. Tm studies were carried out to characterize gRNA-DNA binding. The gRNAs were subsequently tested in solution for their ability to cut dsDNA in the presence of Cas12a. In-solution CRISPR experiments showed that both m5C and m6A modifications improved nuclease activity. The approach can be used for further application for in cell studies by including additional modifications, such as phosphorothioate backbone.

License

Creative Commons Attribution 4.0 International License
This work is licensed under a Creative Commons Attribution 4.0 International License.

Included in

Chemistry Commons

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