University of Cambridge
Focused CRISPR-Cas9 screens investigating the DNA damage response
Abstract
dc:description.abstractThe DNA damage response (DDR) consists of a complex network of interconnected pathways which detect and repair damaged DNA, maintaining genome integrity. Somatic mutations in DDR genes are associated with tumour development, while germline mutations cause a spectrum of disorders ranging from inherited cancer predisposition syndromes to developmental disorders. Genetic screens can be used to gain insights into the function of known DDR proteins, identify novel DDR components and highlight potential therapeutic opportunities. We demonstrate that CRISPR-Cas9 gene editing is a useful tool in genetic screens investigating the DDR. We present the results of three screens using focused CRISPR-Cas9 guide RNA libraries each interrogating different aspects of DDR biology. Firstly, we used a kinase-focused guide RNA library in combination with three clinically relevant DNA damaging agents. We identified kinase knockouts causing sensitivity and resistance to these agents and validated our results in an additional cellular background. These results highlight opportunities for personalised medicine in cancer treatments including potential combinations of kinase inhibitors with more traditional therapeutic DNA damaging agents. In addition, we address key issues in optimal CRISPR-Cas9 screen design, in particular the effect of cellular p53 status on screen sensitivity. Parallel CRISPR-Cas9 screens using a DDR-focused dual guide RNA library in wild-type and TP53 knockout RPE-1 cell lines demonstrate that p53 has a demonstrable impact in reducing screen sensitivity. However, with optimal screen design and high representation we show that biologically relevant targets can be identified in p53 proficient cells. Finally, we present results from a focused CRISPR-Cas9 screen identifying SLFN11 as a novel treatment target in the DNA repair disorder Xeroderma Pigmentosum. We show that SLFN11 depletion rescues ultraviolet (UV) hypersensitivity in cells deficient in nucleotide excision repair and translesion synthesis. We propose that increased cell survival associated with SLFN11 depletion arises from utilisation of a combination of repair pathways in which cells remain proficient, including homologous recombination and the use of alternative translesion polymerases.
Degree
thesis:*- Name dc:type.qualificationname
- Doctor of Philosophy (PhD)
- Level dc:type.qualificationlevel
- Doctoral
- Grantor dc:publisher.institution
- University of Cambridge
- Year dc:date.issued
- 2020
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Bowden, Anne
- Advisor dc:contributor.advisor
-
- Jackson, Stephen
Subjects
dc:subject × 8Rights
dc:rightsIdentifiers
dc:identifier.*- Author Identifier
-
0000-0003-1138-4452
0000-0001-9317-7937 - OAI identifier oai:identifier
- oai:www.repository.cam.ac.uk:1810/321352