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Combinatorial Gene Editing in Human Cells to Model Deficiencies of DNA Repair in Cancer

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Universitat de Barcelona. Facultat de Medicina i Ciències de la Salut

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[eng] DNA repair mechanisms are essential for maintaining genome stability, yet the intricate interactions between various repair pathways remain incompletely understood. Despite the growing recognition of genetic interactions in shaping cellular phenotypes, current combinatorial knockout strategies remain constrained by robustness, scalability, and flexibility, limiting their utility in high-throughput functional genomics. In this doctoral thesis we introduce two highly flexible and cost-efficient approaches for combinatorial knockout generation utilizing CRISPR/Cas12a technology as well as a modular approach for constructing pairwise combinatorial libraries that enable large-scale screening of gene interactions. We applied our method to generate isogenic cell line panels with combinatorial knockouts to systematically interrogate DNA repair genes related to MMR, BER and DR in a pairwise manner. Our results demonstrate high specificity after library cloning, with sequencing validation showing an average of 89% of reads aligning to expected combinations among several library constructions. Despite some challenges in maintaining balanced representation of modules within the libraries, our strategy provides a scalable and efficient solution for multiplexed gene editing. Furthermore, the implementation of dual gene targeting (two distinct crRNAs targeting the same gene) significantly improved editing outcomes with single-gene knockouts achieving 89.87% efficiency and double knockouts reaching 79.08%. Applying our double knockout CRISPR array with dual gene targeting to a human cancer cell line, we characterized the mutational signature profiles accumulated by the knockout clones over the course of two years. With the recovery of the over 1,000,000 mutations accumulated, we observed distinct mutational burdens that clarified the contributions of individual repair genes. Core MMR knockouts (MSH2, MSH6, MLH1, and PMS2) exhibited high mutation accumulation and microsatellite instability (MSI) signatures, consistent with known repair deficiencies. Interestingly, MSH6-deficient cells accrued fewer indels than other MMR knockouts, reinforcing findings that MSH6 loss can be partially compensated by MSH3-mediated repair. Furthermore, we identified a unique mutational signature (SigC) specifically associated with MLH1 deficiency, suggesting potential clinical applications in distinguishing MLH1- and MSH2-deficient tumors. The loss of PMS2 resulted in a distinct mutational profile, which was further exacerbated by the combined loss of MLH3, suggesting compensatory roles of MutLβ (MLH1-MLH3) in MMR-deficient backgrounds. Additionally, we uncovered a potential role for MSH5 in somatic mutagenesis, as the MSH6-MSH5 double knockout displayed an increased mutation burden distinct from either single-gene knockout. These findings challenge the traditional view of MSH5 as meiosis-specific and highlight its possible contribution to somatic DNA repair. Our analysis of small insertions and deletions (indels) in microsatellite regions provides new insights into MSI stratification. We demonstrated that different MMR deficiencies result in distinct MSI profiles, with MSH6 knockouts displaying mononucleotide deletions, while PMS2 knockouts showed a bias toward insertions. Additionally, our results support the association of MSH3 deficiency with Elevated Mutagenic Alterations at Specific Tetranucleotide Repeats (EMAST), resolving an ongoing debate about the genomic presence of EMAST in human cells. The study also revealed a strong interaction between MutSα (MSH2-MSH6) and oxidative damage repair pathways. Our OGG1 knockout data demonstrated a mutational pattern distinct from the known oxidative damage signature (COSMIC SBS18), suggesting that OGG1 deficiency produces a unique mutational footprint. Interestingly, MSH6-OGG1 double knockouts exhibited increased mutagenesis, indicating that MutSα may recognize oxidative damage in vivo, a hypothesis supported by previous in vitro studies, but lacking prior genomic validation. This finding has significant implications for understanding the interplay between mismatch repair and oxidative damage processing. Our statistical framework identified significant epistasis between PMS2 and MLH1, reinforcing the hypothesis that loss of either gene does not completely abrogate MMR activity. Similarly, our model detected a functional interaction between MMR genes and the BER enzyme MUTYH, supporting the idea that MMR contributes to the repair of oxidative lesions, possibly through recruitment of translesion synthesis (TLS) polymerases. This work provides an effective strategy to screen genetic interactions through the use of a flexible, high-throughput dual gene targeting CRISPR array. With this, we have uncovered novel insights into DNA repair pathways, their redundancies, and their mutational consequences. This not only advances our fundamental understanding of DNA repair but also establishes a robust methodological framework for future combinatorial screens.

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ADVERTIMENT. Tots els drets reservats. L'accés als continguts d'aquesta tesi doctoral i la seva utilització ha de respectar els drets de la persona autora. Pot ser utilitzada per a consulta o estudi personal, així com en activitats o materials d'investigació i docència en els termes establerts a l'art. 32 del Text Refós de la Llei de Propietat Intel·lectual (RDL 1/1996). Per altres utilitzacions es requereix l'autorització prèvia i expressa de la persona autora. En qualsevol cas, en la utilització dels seus continguts caldrà indicar de forma clara el nom i cognoms de la persona autora i el títol de la tesi doctoral. No s'autoritza la seva reproducció o altres formes d'explotació efectuades amb finalitats de lucre ni la seva comunicació pública des d'un lloc aliè al servei TDX. Tampoc s'autoritza la presentació del seu contingut en una finestra o marc aliè a TDX (framing). Aquesta reserva de drets afecta tant als continguts de la tesi com als seus resums i índexs.

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Tesis Doctorals - Facultat - Medicina i Ciències de la Salut

# metadata.dc.title[].value
Combinatorial Gene Editing in Human Cells to Model Deficiencies of DNA Repair in Cancer