Millar, Rhona;
(2025)
Arrayed whole-genome CRISPR screen identifies novel regulators of cGAS biology in response to DNA damage.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
Micronuclei play a crucial role in cancer development and progression through large-scale genomic rearrangements. Additionally, micronuclei have been implicated in the induction of cGAS-STING mediated innate immune signalling by serving as a source of self-dsDNA. Activation of which has conflicting implications within cancer cells, potentially driving tumour rejection as well as metastatic progression. In this work, we developed an arrayed whole genome CRISPR Cas9 screening strategy to identify factors which impact micronuclei formation in response to irradiation and factors which regulate subsequent recognition by cGAS. This approach enabled identification of two novel factors involved in cGAS engagement of micronuclei: RNF113A and C11orf73 (Hikeshi). Downregulation of C11orf73, a nuclear transport receptor, resulted in increased cGAS accumulation at micronuclei following irradiation. In contrast, downregulation of the E3 ubiquitin ligase RNF113A led to a reduction in cGAS recruitment to micronuclei arising from both irradiation and chromosome segregation errors. Further investigation revealed that RNF113A downregulation also reduced cGAS localisation at primary nuclei, reduced global cGAS protein levels and attenuated cGAMP production in response to immune-stimulatory DNA (ISD). In addition, we show here that micronuclei alone are insufficient to activate cGAS-STING signalling and subsequent innate immune cytokines. We further demonstrate that histones on self dsDNA derived from micronuclei inhibit cGAS activation, consistent with findings from independent groups. Collectively, these results have implications for oncology treatment strategies that rely on DNA-damage driven immune responses, as well as our fundamental understanding of cGAS-dependent autoimmune pathologies.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Arrayed whole-genome CRISPR screen identifies novel regulators of cGAS biology in response to DNA damage |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Copyright © The Author 2025. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request. |
| Keywords: | Radiation, DNA damage response, Micronuclei |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Cancer Institute UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Cancer Institute > Research Department of Oncology |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10212040 |
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