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.
| Identifer | oai:union.ndltd.org:umassmed.edu/oai:escholarship.umassmed.edu:gsbs_diss-2100 |
| Date | 14 July 2020 |
| Creators | Lee, Jooyoung |
| Publisher | eScholarship@UMassChan |
| Source Sets | University of Massachusetts Medical School |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Morningside Graduate School of Biomedical Sciences Dissertations and Theses |
| Rights | Licensed under a Creative Commons license, http://creativecommons.org/licenses/by/4.0/ |
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