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Protospacer Selection within the CRISPR Mechanism Against Phage Evolution
Department: Biology
ResourceLengthWidthThickness
Paper000
Specimen Elements
Pocatello
Unknown to Unknown
Eva Githuku
Idaho State University
Thesis
No
6/25/2025
digital
City: Pocatello
Master
Due to their life cycles, phages have the potential to be used as an alternative to antibiotics to counter antibacterial resistance. Phage therapy is not yet widespread, partly due to bacterial defense systems, such as CRISPR. To counter CRISPR, phages have developed point mutations within their genomes. This study aims to identify and assess the mutation variations of the protospacer regions within the phage genome. In vitro and computational analysis assessed natural selection in protospacer-containing genes within cluster members of mesophilic and thermophilic phages. The purification of thermophilic phages was unsuccessful, partly due to the specificity of the isolated phages to host receptors. Analysis of protospacer-containing genes in mesophilic phage genomes revealed widespread purifying selection consistent with strong functional constraints on phage proteins, as most genes showed dN/dS < 1. However, positive selection (dN/dS > 1) was observed, indicating that the phage would favor mutations in these regions, used to evade the CRISPR-Cas system. In addition to identifying a suitable host for phage isolation, the microbial diversity analysis provided insight into the role of temperature in shaping microbial communities. 16S rRNA gene sequencing generated 7,618 Amplicon Sequence Variants (ASVs). No significant difference in alpha diversity indices among temperature groups (p > 0.05) was observed. Beta diversity analysis (PERMANOVA, R² = 0.229, p = 0.001) indicated that temperature significantly influenced community structure. Bipartite network plots showed that most ASVs were site-specific, suggesting thermal specialization, while a smaller subset had broader temperature ranges and occurred in multiple sites. Keywords: CRISPR, protospacer-containing genes, dN/dS, purifying selection, ASV, microbial community

Protospacer Selection within the CRISPR Mechanism Against Phage Evolution

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