Engineering Circular Guide RNAs for Enhanced CRISPR-Cas9 Efficiency
| dc.contributor.advisor | Plesa, Calin | |
| dc.contributor.advisor | Raisanen, Elizabeth | |
| dc.contributor.author | Keller, Abigail | |
| dc.date.accessioned | 2024-12-12T22:19:15Z | |
| dc.date.available | 2026-12-12T22:19:15Z | |
| dc.date.issued | 2024-11 | |
| dc.description | 46 pages | |
| dc.description.abstract | The CRISPR-Cas9 system has revolutionized genome editing, offering remarkable precision and versatility in targeted genetic modifications. This study presents an approach to improve CRISPR-Cas9 efficiency through the design and validation of circular guide RNA (cgRNA) constructs. Circular RNAs are more stable than their linear counterparts which are susceptible to degradation by extracellular RNases that are abundant in the environment and act on their ends. Prior studies suggest that effective cgRNAs can be produced from self-splicing Group I introns, but the optimal linker lengths for efficient circularization remain unknown. The linker, essential for connecting the ends of the cgRNA, is an added sequence that prevents tight folding, allowing stable circularization without interfering with the RNA's editing efficiency. Our goal enables the production of stable cgRNAs using a Golden Gate Assembly (GGA) plasmid incorporating a gRNA template and self-splicing group I introns. Following successful validation of our GGA constructs in Escherichia coli cells, numerous cgRNA linkers can be tested to find the optimal linker for cgRNA stability and improved CRISPR efficiency. Circular guide RNAs exhibit enhanced efficiency and stability compared to linear counterparts, thereby providing valuable insights for advancing CRISPR-Cas9 technology and its applications in genome editing. | |
| dc.identifier.orcid | 0009-0003-4854-1631 | |
| dc.identifier.uri | https://hdl.handle.net/1794/30239 | |
| dc.language.iso | en_US | |
| dc.publisher | University of Oregon | en |
| dc.rights | Creative Commons BY-NC-ND 4.0-US | |
| dc.subject | bioengineering, CRISPR Cas9, RNA, RNA circularization, genetic editing | |
| dc.title | Engineering Circular Guide RNAs for Enhanced CRISPR-Cas9 Efficiency | |
| dc.type | Thesis |