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
This work is licensed under a Creative Commons Attribution 4.0 International License.
Recommended Citation
Dagci, Hilal, "Enhancing CRISPR-Cas12a Activity through Guide RNA Methylation" (2025). Electronic Theses & Dissertations (2024 - present). 221.
https://scholarsarchive.library.albany.edu/etd/221
