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Generating an isogenic model of the APOBEC3A_B deletion polymorphism using CRISPR/Cas9

Biondo, Rosalba (2020) Generating an isogenic model of the APOBEC3A_B deletion polymorphism using CRISPR/Cas9. Master of Science by Research (MScRes) thesis, University of Kent,. (KAR id:80472)

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Abstract

Deamination of cytosine, leading to C>T transitions, are one of the most common mutation leading to carcinogenesis. Recently, APOBEC3 (A3) enzymes have been accounted as the main cause. Under normal conditions, these deaminase enzymes have antiviral activities, which mutate viral genome preventing infections. Deregulation of these enzymes has been linked with C>T mutation in host genome, within the specific TpCpW (where W = A or T) nucleotide sequence, termed APOBEC-signature mutations. These are very common in several tumours, such as breast, cervical, head and neck and lung cancer. A3B has predominantly been associated with high APOBEC-signature mutations, especially in breast cancer. However, a naturally occurring ~29.5kb germline deletion polymorphism, spanning from intron 4 of A3A to intron 7 of A3B, generates the hybrid A3A_B, which causes the loss of almost the entire A3B. However, A3B-null tumours still present high APOBEC-signature mutations and have a high cancer risk. To investigate this paradox, we generated an isogenic model, using CRISPR/Cas9, which replicates the ~29.5kb deletion in a non-cancerous cell line. The genotyping assay created is very sensitive as it was possible to detect as low as 1% of cells with the deletion in a heterogeneous population. Furthermore, we analysed the use of the dual puro-ΔTK selection cassette in NIKS, to enrich for cells with the deletion and laid the ground for future studies. This is a novel and important system, which can help understand how A3B deletion might affect the other A3 enzymes and cell proliferation, what drives carcinogenesis in these cells and what are the impacts on treatment development and advances. Overall the results obtained from this work are important for further analysis on APOBEC3 and its implication in cancer formation, prompting further studies and analysis.

Item Type: Thesis (Master of Science by Research (MScRes))
Thesis advisor: Fenton, Tim
Subjects: Q Science > QH Natural history
Divisions: Faculties > Sciences > School of Biosciences
SWORD Depositor: System Moodle
Depositing User: System Moodle
Date Deposited: 16 Mar 2020 12:23 UTC
Last Modified: 17 Mar 2020 08:25 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/80472 (The current URI for this page, for reference purposes)
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