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Cannon, Christopher (2025) Applications of a high-throughput screen detecting CRISPR-Cas spacer acquisition. PhD thesis, University of Nottingham.

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Abstract

The arms race between prokaryotes and their foreign genetic elements has driven the evolution of a diverse and enigmatic array of immune systems. CRISPR-Cas systems uniquely provide adaptive immunity against these foreign elements. Immunity is achieved through the acquisition of DNA fragments derived from invaders’ genomes, catalysed by the Cas1-Cas2 integrase complex. These fragments are stored as spacers within CRISPR arrays and are transcribed to specifically direct the effector machinery against their complementary sequences.

Mutations in the Cas1 protein of the E. coli Type I-E CRISPR-Cas system have been identified that exhibit an increased rate of spacer acquisition. It has not been practical to perform systematic large-scale screening of mutagenic libraries using previous assays. I used a papillation reporter assay system to identify novel hyperactive mutations which exhibit up to five-fold increases in rates of spacer acquisition. These mutants also induce an elevated SOS response, suggesting increased integrase activity has the potential to confer a negative fitness cost to the host cell.

The screening of metagenomic libraries to identify anti-CRISPRs targeting the integrase machinery identified homologs of genes ancillary to the process of spacer acquisition as potential inhibitors. This suggests a mechanism whereby genetic elements may inhibit CRISPR systems without directly targeting the cas genes.

Finally, I constructed and tested several versions of a genetic circuit for the high-throughput detection of spacer acquisition, which may be universally applied to CRISPR systems.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Chalmers, Ronald
Keywords:	Immunity; DNA fragments; Cas1-Cas2 integrase complex; Genetic circuit
Subjects:	Q Science > QR Microbiology > QR180 Immunology
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID:	80674
Depositing User:	Cannon, Christopher
Date Deposited:	24 Jul 2025 04:40
Last Modified:	24 Jul 2025 04:40
URI:	https://eprints.nottingham.ac.uk/id/eprint/80674

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