{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/342218"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/342218","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Harnessing CRISPR-Cas9 screens to identify functional genetic interactions","abstract":"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.","abstract_html":"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.","abstract_has_math":false,"creators":["Morales Juarez, David"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Jackson, Stephen P"],"committee_chairs":[],"committee_members":[],"year":2022,"date_issued":"2022-07-14","date_published":"2022-07-14","updated_at":"2026-07-22T22:24:06Z","subjects":["CRISPR-Cas9","Genetic Screening","DNA Damage Response","Synthetic Lethality","p53","DNA Topoisomerases","WRN","Microsatellite Instability"],"languages":["eng"],"rights":[],"rights_urls":["https://www.rioxx.net/licenses/all-rights-reserved/"],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.89637","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Jackson, Stephen P"]},{"key":"dc:contributor.sponsor","label":"Sponsor","values":["CONACYT Cambridge Scholarship"]},{"key":"dc:creator","label":"Author","values":["Morales Juarez, David"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2022-07-14"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/342218"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["CRISPR-Cas9","Genetic Screening","DNA Damage Response","Synthetic Lethality","p53","DNA Topoisomerases","WRN","Microsatellite Instability"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["https://www.rioxx.net/licenses/all-rights-reserved/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["10.17863/CAM.89637"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/311648da-99eb-4c6d-8010-892c60ff6f1d/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["The advent of CRISPR-Cas9 genetic engineering technologies has vastly improved our ability to interrogate genetic interactions in high-throughput studies. 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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."]},{"key":"dc:format.checksum.md5","label":"Dc Format Checksum Md5","values":["8f8ff61463d75a8d426271c12b003818"]},{"key":"dc:title","label":"Title","values":["Harnessing CRISPR-Cas9 screens to identify functional genetic interactions"]}]}],"canonical_facts":{"dc:contributor.advisor":["Jackson, Stephen P"],"dc:contributor.sponsor":["CONACYT Cambridge Scholarship"],"dc:creator":["Morales Juarez, David"],"dc:date.issued":["2022-07-14"],"dc:description.abstract":["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. 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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."],"dc:format.checksum.md5":["8f8ff61463d75a8d426271c12b003818"],"dc:identifier.doi":["10.17863/CAM.89637"],"dc:identifier.uri":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/311648da-99eb-4c6d-8010-892c60ff6f1d/download"],"dc:language":["eng"],"dc:publisher.institution":["University of Cambridge"],"dc:relation.isreferencedby.uri":["https://www.repository.cam.ac.uk/handle/1810/342218"],"dc:rights":["https://www.rioxx.net/licenses/all-rights-reserved/"],"dc:subject":["CRISPR-Cas9","Genetic Screening","DNA Damage Response","Synthetic Lethality","p53","DNA Topoisomerases","WRN","Microsatellite Instability"],"dc:title":["Harnessing CRISPR-Cas9 screens to identify functional genetic interactions"],"dc:type":["Thesis"],"dc:type.qualificationlevel":["Doctoral"],"dc:type.qualificationname":["Doctor of Philosophy (PhD)"]},"updated_at":"2026-07-22T22:24:06Z"}