Development of a high-throughput screen for the study of CRISPR spacer acquisition

Braithwaite, Jack (2021) Development of a high-throughput screen for the study of CRISPR spacer acquisition. PhD thesis, University of Nottingham.

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Bacteria are in an ever-evolving arms race to survive against invasion from foreign genetic elements. One mechanism of defence is the CRISPR-Cas system of adaptive immunity. Through CRISPR-Cas, cells store memories of previous invaders as DNA fragments, or spacers, in a genomic array of repeats. Once transcribed, these spacers guide CRISPR associated (Cas) proteins in the sequence-specific destruction of matching invaders.

Much of the CRISPR-Cas immune response is well understood, owing to the close genetic linkage between the array and the genes that facilitate immunity. However, elements of spacer capture and integration by Cas1 and Cas2 proteins remain elusive. This is partly due to the absolute requirement of distantly related host factors that aid in diverse processes, including the generation of novel spacers and their integration into arrays.

Thus far, discovery of these host factor genes has been limited by the hypothesis driven interrogation of bacterial genomes. The necessity of such an approach is a result of the dependence on single throughput genetic screening. In this work, I present a novel assay for the high-throughput detection of spacer integration. This assay can uniquely detect E.coli spacer acquisition in plated colonies to a high degree of sensitivity. Unlike other detection methods, this assay can support the discovery of host factor genes through random genetic screening.

Using this assay, I screened 15,000 transposon insertion mutants for spacer acquisition activity in search of novel host factor genes. This led to the isolation of 59 hypo-active mutants. Sequencing of transposon insertion sites revealed 29 individual genes with a possible role in spacer acquisition.

Subsequent bioinformatic analysis implicated a vast array of cellular processes in spacer acquisition, from intracellular signalling to DNA repair pathways. Most significantly, these results suggest that spacer acquisition in E.coli is dependent on cell filamentation.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Chalmers, Ronald
Allers, Thorsten
Keywords: High-throughput screen, CRISPR, Spacer acquisition
Subjects: Q Science > QR Microbiology > QR180 Immunology
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID: 66912
Depositing User: BRAITHWAITE, Jack
Date Deposited: 18 Jan 2022 14:40
Last Modified: 08 Dec 2023 04:30

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