CRISPR-Cas is an adaptive immune system unique to prokaryotes, which prevents infection by foreign genetic elements. Key to the function of CRISPR-Cas immunity is the ability to adapt to new threats in incorporating short segments, termed spacers, of invading DNA into the clustered regularly interspaced short palindromic repeat (CRISPR) array of the host. Spacers constitute immunological memories, used by CRISPR-associated (Cas) proteins to mount a sequence-specific attack on subsequent infections. The immunisation of the host is called CRISPR adaption. Adaption requires the integration of new spacers at a precise site in the CRISPR array. Two proteins, Cas1 and Cas2, are essential for adaptation; however, the mechanisms of spacer integration remain poorly understood. The work described here focused on understanding adaptation in Sulfolobus solfataricus. Using biochemical assays, I aimed to characterise the activity of the Cas1 and Cas2 proteins in this organism in order to understand their role in the insertion of new spacers. Additionally, I aimed to investigate how the expression of CRISPR-Cas components is regulated in this organism in response to viral infection. The results presented here show that expression of Cas1 was strongly upregulated in response to infection. A Csa3 protein from S. solfataricus was found to bind to the promoter for transcription of cas1, implying a role in the regulation observed. I reconstituted in vitro both the integration reaction performed by Cas1 and Cas2 proteins of S. solfataricus and the reverse of this reaction, disintegration. Cas1 was shown to impose sequence specificity on these reactions, selecting sites similar to the leader-repeat junction of the CRISPR locus. Finally, I demonstrated that, in addition to the intrinsic specificity of Cas1, there was a requirement for an additional host factor for site-specific integration in S. solfataricus.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:730683 |
| Date | January 2017 |
| Creators | Rollie, Clare |
| Contributors | White, Malcolm F. |
| Publisher | University of St Andrews |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/10023/10814 |
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