Zhou, Wenyuan
(2022)
Optical Control of CRISPR/Cas9 and Kinase Function.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Genetic code expansion provides a unique solution by allowing for site-specific installation of functionally masked unnatural amino acids (UAAs) into proteins, rendering them inert until rapid activation of protein function through exposure to light. To facilitate the use of unnatural amino acid mutagenesis in mammalian cells, I will present a comparative evaluation of select plasmid-based genetic code expansion systems and a detailed analysis of suppression efficiency with different UAAs and in different cell lines.
Light has been widely harnessed as a rapid, non-invasive, dose-dependent, and spatiotemporally specific trigger for the regulation of complex biological processes. Oligonucleotides containing photocaged nucleobases abolish hybridization to their complementary strands via disrupted Watson-Crick base pairing, which enables light activation of target recognition and genomic modification through gene editing modalities. In this dissertation, I hereby describe the use of photocaged RNAs to control the interaction between CRISPR/Cas9 and its cognate DNA, which confers optical control of (1) targeted DNA cleavage for gene recombination and gene silencing and (2) targeted DNA binding for transcription activation and base editing.
Moreover, I will leverage a genetically encoded, light-activatable lysine derivative to optically control the activity of (1) an adenylate kinase to regulate ATP levels in both E. coli and mammalian cells, (2) mammalian kinases involved in signal transduction pathways (i.e., the MEK/ERK pathway and the MKK6/p38 pathway) in an isoform-specific fashion, for better understanding of their divergent roles in different biological processes.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
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| Date: |
13 August 2022 |
| Date Type: |
Publication |
| Defense Date: |
16 April 2021 |
| Approval Date: |
4 December 2024 |
| Submission Date: |
3 August 2022 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
295 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
Dietrich School of Arts and Sciences > Chemistry |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
Gene editing, optical control, unnatural amino acid, genetic code expansion, kinase, signaling |
| Date Deposited: |
04 Dec 2024 16:57 |
| Last Modified: |
04 Dec 2024 18:08 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/43486 |
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