Generating Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) gene deletions in Bladder Cancer Cell Lines

Bladder cancer was responsible for 200,000 deaths worldwide in 2018, it has a well-documented ability to evade and resist chemotherapy treatment. Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) enzymes are a family of enzymes and form part of the innate immune system in human cells where they mutate cDNA from infecting viruses. 2 APOBECs: APOBEC3A (A3A) and APOBEC3B (A3B) have been linked not only to bladder cancer, but to half of all cancers. These APOBECs leave a distinct mutational signature which has led to many computational studies into APOBEC involvement in cancers. APOBECs seem to have a direct role in evading chemotheraputic agents; through upregulation in cancers and increased mutational signature after rounds of chemotherapy. Because of this it has been postulated that development of techniques to downregulate APOBEC expression in cancers, alongside traditional chemotherapy drugs, will stop recurrence. To test this theory cancer-cell lines with homozygous knockouts for A3A and/or A3B must be created to assess the link between these genes and resistance to traditional chemotherapy drugs. This thesis attempts to create these knockouts in BFTC-905, T24 and 5637 bladder cancer cell lines. A3A was successfully knocked out in one line: BFTC-A2-F, and A3B was potentially knocked out in BFTC-Both3-F and BFTC-Both4-F. This experiment has laid the foundation for more A3A and A3B knockouts to be cloned from pools of knock-out targeted cell lines: 5637, T24 and BFTC-905 or through BFTC-A2-F which can be used as a platform to attempt to knock out A3B too. It would appear that trying to knock-out both A3A and A3B at the same time creates some unexpected results, possibly due to how close these genes are together (~30kb) in the APOBEC3 locus. Deletion of A3B appears hard to achieve, as only a heterozygous deletion in BFTC-B5-F was achieved, these results echo a previous experiment in a normal cell line.