Investigating DNA replication perturbance induced by CRISPR-Cas9 variants
Single-Stranded DNA breaks (SSBs) represent one of the most abundant DNA lesions occurring in the cells of living organisms. If encountered by the DNA replication machinery, SSBs can be converted into double-stranded DNA breaks (DSBs). Homologous recombination (HR) pathways are deployed to repair th...
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| Format: | Thesis |
| Language: | English |
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2023
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| _version_ | 1826312511125716992 |
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| author | Xu, Y |
| author2 | Whitby, M |
| author_facet | Whitby, M Xu, Y |
| author_sort | Xu, Y |
| collection | OXFORD |
| description | Single-Stranded DNA breaks (SSBs) represent one of the most abundant DNA lesions occurring in the cells of living organisms. If encountered by the DNA replication machinery, SSBs can be converted into double-stranded DNA breaks (DSBs). Homologous recombination (HR) pathways are deployed to repair these DSBs and initiate replication restart. However, HR-mediated replication restart, whilst helping to ensure that DNA replication is completed prior to cell division, is a potential cause of genome instability due to the possibility of recombination between ectopic homologous sequences, and the compromised stability and fidelity of the restarted replication fork.
In this study, I have established a site- and strand-specific nicking system using CRISPR-Cas9 nicking variants (Cas9n) to investigate the formation and repair of replication associated DSBs in fission yeast. I discovered that SSBs induced by Cas9n in the leading/lagging strand template are converted into two-ended DSBs upon concerted replication fork stalling and fork convergence at the SSB. The Cas9n induced replication associated DSBs give arise to high levels of direct repeat recombination through Rad51 mediated Synthesis Dependent Strand Annealing (SDSA)/Sister Chromatid Exchange (SCE) and Rad52 mediated Single Strand Annealing (SSA). I have also shown that Cas9n induced SSBs in both leading and lagging strand templates can trigger Break-Induced Replication (BIR) and associated template switching downstream.
Other than SSBs, DNA-protein complexes are also proposed to be major impediments to ongoing DNA replication. Examples of such barriers include the programmed DNA-protein barrier RTS1 and non-programmed DNA-protein barriers such as lacO-LacI. To further investigate what happens when replication forks encounter DNA-protein complexes in fission yeast, I used catalytically dead Cas9 (Cas9d) to create a site-specific barrier. As the Cas9d-DNA complex contains an R-loop, it may mimic other R-loop containing protein complexes such as the transcription machinery and, therefore, provide a model for better understanding of transcription-replication conflicts. I found that Cas9d is only a weak barrier to DNA replication in fission yeast, yet it can strongly induce direct repeat recombination that is primarily driven by Rad51. Intriguingly, this recombination is restricted to ~2kb region adjacent to the gRNA binding site and does not lead to any recombination dependent replication (RDR) associated template switching downstream of the gRNA site. These features distinguish Cas9d from the RTS1 replication fork barrier, which causes a wider spread of recombination activity adjacent to it and high levels of template switching downstream.
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| first_indexed | 2024-04-09T03:55:41Z |
| format | Thesis |
| id | oxford-uuid:feae6bc1-5b57-4f26-bdc1-37708a60ec48 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-04-09T03:55:41Z |
| publishDate | 2023 |
| record_format | dspace |
| spelling | oxford-uuid:feae6bc1-5b57-4f26-bdc1-37708a60ec482024-03-08T12:49:38ZInvestigating DNA replication perturbance induced by CRISPR-Cas9 variantsThesishttp://purl.org/coar/resource_type/c_db06uuid:feae6bc1-5b57-4f26-bdc1-37708a60ec48Molecular geneticsBiochemistryEnglishHyrax Deposit2023Xu, YWhitby, MSingle-Stranded DNA breaks (SSBs) represent one of the most abundant DNA lesions occurring in the cells of living organisms. If encountered by the DNA replication machinery, SSBs can be converted into double-stranded DNA breaks (DSBs). Homologous recombination (HR) pathways are deployed to repair these DSBs and initiate replication restart. However, HR-mediated replication restart, whilst helping to ensure that DNA replication is completed prior to cell division, is a potential cause of genome instability due to the possibility of recombination between ectopic homologous sequences, and the compromised stability and fidelity of the restarted replication fork. In this study, I have established a site- and strand-specific nicking system using CRISPR-Cas9 nicking variants (Cas9n) to investigate the formation and repair of replication associated DSBs in fission yeast. I discovered that SSBs induced by Cas9n in the leading/lagging strand template are converted into two-ended DSBs upon concerted replication fork stalling and fork convergence at the SSB. The Cas9n induced replication associated DSBs give arise to high levels of direct repeat recombination through Rad51 mediated Synthesis Dependent Strand Annealing (SDSA)/Sister Chromatid Exchange (SCE) and Rad52 mediated Single Strand Annealing (SSA). I have also shown that Cas9n induced SSBs in both leading and lagging strand templates can trigger Break-Induced Replication (BIR) and associated template switching downstream. Other than SSBs, DNA-protein complexes are also proposed to be major impediments to ongoing DNA replication. Examples of such barriers include the programmed DNA-protein barrier RTS1 and non-programmed DNA-protein barriers such as lacO-LacI. To further investigate what happens when replication forks encounter DNA-protein complexes in fission yeast, I used catalytically dead Cas9 (Cas9d) to create a site-specific barrier. As the Cas9d-DNA complex contains an R-loop, it may mimic other R-loop containing protein complexes such as the transcription machinery and, therefore, provide a model for better understanding of transcription-replication conflicts. I found that Cas9d is only a weak barrier to DNA replication in fission yeast, yet it can strongly induce direct repeat recombination that is primarily driven by Rad51. Intriguingly, this recombination is restricted to ~2kb region adjacent to the gRNA binding site and does not lead to any recombination dependent replication (RDR) associated template switching downstream of the gRNA site. These features distinguish Cas9d from the RTS1 replication fork barrier, which causes a wider spread of recombination activity adjacent to it and high levels of template switching downstream. |
| spellingShingle | Molecular genetics Biochemistry Xu, Y Investigating DNA replication perturbance induced by CRISPR-Cas9 variants |
| title | Investigating DNA replication perturbance induced by CRISPR-Cas9 variants |
| title_full | Investigating DNA replication perturbance induced by CRISPR-Cas9 variants |
| title_fullStr | Investigating DNA replication perturbance induced by CRISPR-Cas9 variants |
| title_full_unstemmed | Investigating DNA replication perturbance induced by CRISPR-Cas9 variants |
| title_short | Investigating DNA replication perturbance induced by CRISPR-Cas9 variants |
| title_sort | investigating dna replication perturbance induced by crispr cas9 variants |
| topic | Molecular genetics Biochemistry |
| work_keys_str_mv | AT xuy investigatingdnareplicationperturbanceinducedbycrisprcas9variants |