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Thesis
Uncovering novel TMEJ factors: a CRISPR-based approach
Master of Science (M.S.), Drexel University
Aug 2025
DOI:
https://doi.org/10.17918/00011126
Abstract
Mutations in the BRCA1 gene are among the most common causes of hereditary breast and ovarian cancers. BRCA1 encodes a protein essential for the high-fidelity repair of DNA double-strand breaks (DSBs) via homologous recombination (HR). In BRCA1-deficient cells, HR is impaired resulting in an increased reliance on error-prone repair pathways such as non-homologous end joining (NHEJ) and DNA polymerase theta-mediated end joining (TMEJ). DNA polymerase theta (Pol[theta]) facilitates TMEJ by promoting the synapsis and fill in synthesis at DSBs containing 3' single-stranded DNA overhangs using short microhomologies to align DNA ends and initiate repair. Pol[theta] inhibition has been shown to selectively kill BRCA1-mutant cells, making it an emerging therapeutic strategy. Relative to other DSB repair pathways, TMEJ and the proteins that regulate this pathway are not well understood. Thus, we sought to identify novel factors that contribute to the TMEJ repair process. To achieve this, we conducted a genome-wide CRISPR-Cas9 knockout screen in Brca1-wild-type and mutant mouse embryonic fibroblasts (MEFs). The screen revealed several genes that reduced cell viability in TMEJ-dependent cells. We further validated these candidates using a sulforhodamine B (SRB) growth assay and identified one gene, Rabl3, with a particularly strong impact on cell viability. However, siRNA-mediated knockdown of Rabl3 did not reduce the cell viability of Brca1 mutant cells. Interestingly, Rabl3 is chromosomally located adjacent to the Polq gene, potentially indicating that genetic disruption of Rabl3 impacts Polq gene regulation in a broader locus regulatory network. This possibility will be subject to future studies exploring this locus and how its genetic regulation impacts TMEJ. This work highlights the importance of rigorous validation of CRISPR screen hits and the potential influence of the broader Polq gene locus as a regulator of DSB repair.
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Details
- Title
- Uncovering novel TMEJ factors
- Creators
- George Frederick Joseph
- Contributors
- Neil Johnson (Advisor)Christian Sell (Advisor)
- Awarding Institution
- Drexel University
- Degree Awarded
- Master of Science (M.S.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- 66 pages
- Resource Type
- Thesis
- Language
- English
- Academic Unit
- Biochemistry and Molecular Biology; College of Medicine; Drexel University
- Other Identifier
- 991022074328304721