Enabling high-efficiency precision genome editing and writing through coupling of natural retron elements
Abstract/Contents
- Abstract
- CRISPR has revolutionized genome editing, but it remains challenging to incorporate desired edits with high-efficiency. Recently, a CRISPR-based technology named Cas9-Retron precISe Parallel Editing via homologY (CRISPEY) has precisely introduced thousands of specific genetic variants with each cell receiving one variant edit in a single experiment by facilitating homology-directed repair (HDR) using retrons, which are bacterial genetic elements that can produce an abundance of multicopy single-stranded DNA (msDNA) to act as donor DNA via RNA reverse transcription. However, CRISPEY is still limited by target site choices, compatible retron options, and the incorporation of longer insert lengths. To improve this system, my thesis tests the ability of a CRISPR-compatible Cas9 variant, xCas9 to cleave at NGA and NGT PAM sites, the ability of Myxococcus xanthus (Mx) retrons to produce msDNA as donor DNA to facilitate HDR, and the ability of an E. coli retron to incorporate the insertion of the NatMX6 gene in yeast cells. xCas9 showed a substantial amount of editing at NGA and NGT. The Mx retrons displayed 9% greater editing efficiency than the Ec86 retron used in the original CRISPEY construct. For the NatMX6 gene insertion, 96.9% of the cells contained the proper edit. These findings suggest a Cas9 variant that can expand target space while maintaining its functionality for the NGG PAM site is still needed. Meanwhile, the Mx retrons are shown to be promising bacterial retrons in improving editing efficiency. Additionally, the NatMX6 gene insertion showed CRISPEY has promising potential for genome writing. Improving CRISPEY will advance gene editing applications to better understand genetic outcomes and effectively develop treatments for genetic disorders.
Description
| Type of resource | text |
|---|---|
| Date modified | December 5, 2022 |
| Publication date | May 6, 2022; May 2022 |
Creators/Contributors
| Author | Vu, Amberly |
|---|---|
| Thesis advisor | Fraser, Hunter |
| Thesis advisor | Qi, Stanley |
| Degree granting institution | Stanford University, Department of Biology |
Subjects
| Subject | Biology, Molecular Genetics, CRISPR, Cas9, Retrons, Genome Editing |
|---|---|
| Genre | Text |
| Genre | Thesis |
Bibliographic information
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- Use and reproduction
- User agrees that, where applicable, content will not be used to identify or to otherwise infringe the privacy or confidentiality rights of individuals. Content distributed via the Stanford Digital Repository may be subject to additional license and use restrictions applied by the depositor.
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 4.0 International license (CC BY-NC).
Preferred citation
- Preferred citation
- Vu, A. and Fraser, H. (2022). Enabling high-efficiency precision genome editing and writing through coupling of natural retron elements. Stanford Digital Repository. Available at https://purl.stanford.edu/kv463ms5298
Collection
Undergraduate Theses, Department of Biology, 2021-2022
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- amberly@stanford.edu
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