2026-09-21T09:09:06Zhttps://api.repository.cam.ac.uk/server/oai/request

oai:www.repository.cam.ac.uk:1810/3971422026-03-24T01:41:11Zcom_1810_307305com_1810_256062col_1810_346969

Epigenetic Determinants of CRISPR-Cas9 genotoxicity https://doi.org/10.17863/CAM.126326 Stalker, Eve Willis, Anne Gene editing therapies, such as those using CRISPR-Cas9, offer promising treatment options for genetic disorders. However, they also present unique safety assessment challenges including detection of off-target effects and unintended genomic alterations. An unresearched area of these concerns involves the role of epigenetic features in influencing Cas9-mediated toxicity. Epigenetics is the study of inheritable changes in gene expression that occur without altering the underlying DNA sequence. Cas9 interacts directly with the genome to introduce double strand breaks at targeted locations. However, the efficiency and specificity of Cas9- mediated editing can be significantly influenced by the epigenetic landscape of the target region. The epigenome shapes chromatin structure determines DNA accessibility, and influences DNA repair processes. These factors could modulate Cas9 activity and the likelihood of off-target effects or chromosomal rearrangements. However, these events are rare enough to be beyond the sensitivity of standard sequencing technologies, so their identification requires bespoke approaches. To assess genotoxicity linked to Cas9-mediated genome editing, the first tool implemented is the Multi-Endpoint Genotoxicity Assessment (MEGA) screen, a high-throughput imaging-based platform designed to identify multiple genotoxicity endpoints. These endpoints can be quantified and provide an assessment of how genotoxicity is impacted. The inclusion of the DNA methyltransferase inhibitor RG108 alongside Cas9 editing allows investigation into how DNA methylation influences genotoxic outcomes following genome editing. Whilst imaging provides a low-cost and standardised method of characterising genotoxicity it lacks the resolution to identify the impacts at a nucleotide level. The genotoxic markers can provide a general idea of what is occurring within the cell, but as it relies on proxies for DSBs and chromosomal damage the direct impact may be missed. To further dissect the relationship between epigenetic features and Cas9-mediated DNA damage, bespoke sequencing technologies were employed. DSBs resulting from genome editing are precisely mapped using the INDUCE-seq method, which labels and quantifies unrepaired DSBs in situ. This technology allows unbiased genome-wide detection of the DSBs formed by Cas9 activity. Chromosomal aberrations, including translocations arising from both on-target and off-target Cas9 activity, are identified using CAST-seq (Chromosomal Aberration Analysis by Single Targeted Ligation-Mediated PCR Sequencing). By integrating ENCODE consortium datasets, the overlap between Cas9-induced translocations and epigenetic features such as open chromatin was characterised in K-562 and HepG2 cell lines. These analyses revealed that regions of open chromatin, typically marked by active histone modifications and open chromatin, are enriched for both offvi target Cas9 binding and chromosomal rearrangements, highlighting the importance of the epigenomic context in predicting and mitigating genotoxic risks during genome editing. University of Cambridge 2025-09-29 Thesis Doctoral Doctor of Philosophy (PhD) eng https://www.repository.cam.ac.uk/handle/1810/397142 embargo 2027-03-23 https://www.repository.cam.ac.uk/bitstreams/53b694bf-f0dc-4a4f-88b7-1b2629799cc3/download e4c31c60851a54f048509065d6bc1028 https://www.repository.cam.ac.uk/bitstreams/b89f6542-e3fc-49bb-b1b1-11892033c91c/download 87eda9de84448d1f82354d60eee3eb5f http://purl.org/NET/rdflicense/allrightsreserved Genotoxicity