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Harnessing CRISPR-Cas9 screens to identify functional genetic interactions 10.17863/CAM.89637 Morales Juarez, David Jackson, Stephen P The advent of CRISPR-Cas9 genetic engineering technologies has vastly improved our ability to interrogate genetic interactions in high-throughput studies. CRISPR-Cas9 screens have rapidly evolved to become the main approach for unbiased, genome-wide, forward genetic studies. This work seeks to harness the innate potential of CRISPR-Cas9 screens to identify functional genetic interactions. First, we clarified the role of the master tumour suppressor, p53, in the feasibility of CRISPR-Cas9 screening by performing parallel focused screens in wild-type and TP53KO human RPE-1 cells and conducting downstream analysis of screen performance. Our results demonstrated that functional p53 negatively impacts the sensitivity of CRISPR-Cas9 screens. Moreover, we established other important factors that affect screen sensitivity, including appropriate guide-RNA representation, sufficient sequencing depth, and the use of multiple high-editing clones. Careful consideration of these factors in screen design and execution allows successful CRISPR-Cas9 screens to be carried out in both p53-proficient and p53-deficient cells, thereby fostering new biological insights. Second, using focused and whole-genome CRISPR-Cas9 screens we identified RAD54L2 as a novel factor involved in cellular responses to the topoisomerase II (TOP2) poison etoposide. Through downstream biochemical studies, we demonstrated that RAD54L2 can counter TOP2-DNA genotoxic adducts, ultimately preventing DNA double-strand break formation and adding a layer of intricacy to the cellular mechanisms that safeguard the genome from TOP2-mediated DNA damage. Since etoposide is widely used for cancer chemotherapy, these findings suggest RAD54L2-mediated resolution of TOP2-DNA adducts could represent a mechanism for tumour adaptation and, therefore, raise RAD54L2 as a potential biomarker for chemotherapy and an attractive candidate for drug discovery. Third, we performed whole-genome CRISPR-Cas9 screens in cancer models of microsatellite instability (MSI) and made headway into identifying novel genetic interactions that can drive resistance or hypersensitivity to WRN-depletion. This work amplifies the evidence for WRN-depletion as a promising new avenue for chemotherapy in cancer models with MSI. Altogether, this work demonstrates the potential of CRISPR-Cas9 screening technologies in identifying functional genetic interactions in a variety of cellular contexts. Additionally, this work contributes to the growing fields of p53 and TOP2 biology and highlights the potential of WRN inhibition as a promising chemotherapeutic agent. University of Cambridge 2022-07-14 Thesis Doctoral Doctor of Philosophy (PhD) eng CONACYT Cambridge Scholarship https://www.repository.cam.ac.uk/handle/1810/342218 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/311648da-99eb-4c6d-8010-892c60ff6f1d/download 8f8ff61463d75a8d426271c12b003818 https://www.rioxx.net/licenses/all-rights-reserved/ CRISPR-Cas9 Genetic Screening DNA Damage Response Synthetic Lethality p53 DNA Topoisomerases WRN Microsatellite Instability