Date of Award

Fall 12-2023

Document Type

Campus Access Thesis

Degree Name

Master of Science (MS)



First Advisor

Michael P. Shiaris

Second Advisor

Katherine Gibson

Third Advisor

Brook T. Moyers


CRISPR-cas systems are an adaptive subset of prokaryotic genomic defenses against viral infection. The systems adapt to novel infections by incorporating short DNA sequences called spacers homologous to the infectious agent into an array between identical segments of palindromic repeats of host DNA. The CRISPR array serves as a memory bank that clears active infections in tandem with compatible cas genes by recognizing and degrading invasive DNA present in the cell. This research focuses on Enterococcus faecalis, a medically significant Gram-positive bacterium present in soils and waters and as a common intestinal commensal of a wide range of animal hosts. It readily undergoes horizontal gene transfer, and mobile genetic elements drive much of the great genetic diversity of the species. Enterococcus faecalis genomes may lack or possess as many as three similar but distinct CRISPR loci in conserved locations on the chromosome. Functional E. faecalis CRISPR-cas loci have been inversely correlated with acquired antibiotic resistance from mobile genetic elements; this is consistent with the capacity to recognize and target invasive DNA. However, the conservation of CRISPR array repeats and distributions of CRISPR loci in E. faecalis populations suggest that more complex interactions between CRISPR variants exist. This research characterizes E. faecalis strains by CRISPR genotype and compares them by isolate source, mobile genetic element carriage, and repeat conservation of CRISPR loci to determine dynamics that may introduce ecological benefits or fitness costs to possessing or lacking specific CRISPR loci. The evidence presented in this work supports ecologically relevant effects of CRISPR-cas genotype, as well as suggests that the uncommon genotype possessing all three loci represents a pre-industrial wild type. CRISPR genotype variation in E. faecalis thus appears to be the result of locus loss through selective pressures and disuse rather than locus acquisition through horizontal gene transfer.


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