Authors
Lee, JooyoungFaculty Advisor
Erik SontheimerAcademic Program
Interdisciplinary Graduate ProgramUMass Chan Affiliations
RNA Therapeutics InstituteDocument Type
Doctoral DissertationPublication Date
2020-07-14Keywords
CRISPRanti-CRISPR
genome engineering
gene editing
Biotechnology
Genetics and Genomics
Microbiology
Other Biomedical Engineering and Bioengineering
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Clustered, regularly interspaced, short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) constitute a bacterial and archaeal adaptive immune system. The ongoing arms race between prokaryotic hosts and their invaders such as phages led to the emergence of anti-CRISPR proteins as countermeasures against the potent antiviral defense. Since the first examples of anti-CRISPRs were shown in a subset of CRISPR-Cas systems, we endeavored to uncover these naturally-occurring inhibitors that inactivate different types of CRISPR-Cas systems. In the first part of my thesis, we have identified and characterized Type II anti-CRISPR proteins that inactivate several Cas9 orthologs. We share mechanistic insights into anti-CRISPR inhibition and show evidence of its potential utility as an off-switch for Cas9-mediated mammalian genome editing. Although the RNA programmability of Cas9 enables facile genetic manipulation with great potential for biotechnology and therapeutics, limitations and safety issues remain. The advent of anti-CRISPR proteins presents opportunities to exploit the inhibitors to exert temporal, conditional, or spatial control over CRISPR. In the second part of my thesis, we demonstrate that anti-CRISPR proteins can serve as useful tools for Cas9 genome editing. In particular, we have demonstrated that anti-CRISPRs are effective as genome editing off-switches in the tissues of adult mammals, and we further engineered anti-CRISPR proteins to achieve tissue-specific editing in vivo. Taken together, my thesis research aimed to mine for natural anti-CRISPR protein inhibitors and repurpose these proteins to complement current Cas9 technologies in basic and clinical research.DOI
10.13028/xxj4-6a50Permanent Link to this Item
http://hdl.handle.net/20.500.14038/31317Rights
Licensed under a Creative Commons licenseDistribution License
http://creativecommons.org/licenses/by/4.0/ae974a485f413a2113503eed53cd6c53
10.13028/xxj4-6a50
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