Jaunky, Brandon Boodhai (2023) Shortening the Half-Life of the CRISPR/dCas9 System. Masters thesis, Concordia University.
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Abstract
Shortening the half-life of genetic engineering tools such as dCas9-VP64 can become a pivotal component to regulate many cellular networks, allowing periodic gene expression and protein levels, thereby governing vital cellular processes. However, currently this effector protein has a prolonged half-life in cells and can cause adverse effects by binding to other regions or interacting with other transcriptional or translational machinery. This thesis aims to explore the N-End Rule as a novel approach to achieve shorter half-life dCas9-VP64, offering tighter control for future applications in synthetic circuits.
The need for shorter half-life gene effectors arises from the cytotoxic effects observed with persistence exposure that can lead to off-target effects and unintended modifications, hampering their accuracy and reliability in genetic manipulation. The introduction of short-lived effector proteins also holds significant promise for enhancing temporal control and predictable timing of protein degradation.
The central hypothesis driving this research is that the N-End Rule can be harnessed to shorten the half-life of the artificial transcriptional activator dCas9-VP64 (Varshavsky, 1998). Through site-directed mutagenesis, four amino acids were introduced at the N-terminus of dCas9-VP64, including alanine, valine, serine, and cysteine. These amino acid residues were identified as destabilizing when positioned at N-termini based on the N-acetylation N-End Rule (Nguyen, K.T. et al., 2018).
Experimental design involved genetic construct development, fusion with a fluorescent protein tag for visualization, and validation of N-terminus mutations. Using cycloheximide, a protein synthesis and translation inhibitor, the steady-state level of the dCas9-VP64-eGFP protein was monitored through eGFP fluorescence from a microplate reader assay and through western blot analysis. We observed different phenotypes during induction and tracking of fusion proteins' signals. Protein half-life was measured using the first order rate kinetic equation for protein degradation.
The results revealed the differential half-life of dCas9-VP64 variants, showcasing the potential of the N-End Rule in achieving shorter half-life. The cysteine variant demonstrated the shortest half-life of 37 minutes, indicating its potential suitability for synthetic circuits. dCas9-VP64-eGFP with the wild-type N-terminus exhibited a half-life of 57 minutes, while serine and alanine variants displayed approximately 54 and 61 minutes, respectively.
In conclusion, this thesis contributes to the advancement of strategies for shortening the half-life of proteins. The implementation of the N-End Rule to achieve this goal exemplifies its potential in optimizing genetic behavior control in future applications with synthetic circuits. By harnessing the N-End Rule, researchers can pave the way for future innovations and advancements in the realm of genetic engineering and molecular biology.
| Divisions: | Concordia University > Faculty of Arts and Science > Biology |
|---|---|
| Item Type: | Thesis (Masters) |
| Authors: | Jaunky, Brandon Boodhai |
| Institution: | Concordia University |
| Degree Name: | M. Sc. |
| Program: | Biology |
| Date: | 17 August 2023 |
| Thesis Supervisor(s): | Zerges, William and Kharma, Nawwaf |
| Keywords: | CRISPR/dCas9, VP64, N-End Rule, Half-Life |
| ID Code: | 992960 |
| Deposited By: | Brandon Jaunky |
| Deposited On: | 14 Nov 2023 19:20 |
| Last Modified: | 14 Nov 2023 19:20 |
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