Browsing by Author "Wiegand, Tanner Roy"

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Intersection of SARS-CoV-2 and CRISPR-CAS defense systems
(Montana State University - Bozeman, College of Agriculture, 2021) Wiegand, Tanner Roy; Chairperson, Graduate Committee: Blake Wiedenheft; This is a manuscript style paper that includes co-authored chapters.
Viral predators exploit cellular resources in all domains of life. To defend against these genetic invaders, bacteria and archaea have evolved adaptive immune systems comprised of clustered regularly interspaced short palindromic repeats (CRISPR) and their associated Cas proteins. In this dissertation, I investigate the biological mechanisms and biotechnological applications of CRISPR-Cas systems. The sequences that interspace the eponymous repeats of CRISPR loci are derived from mobile genetic elements, including bacteriophages (i.e., viruses that infect bacteria). When the locus is transcribed into CRISPR-RNA, these spacer sequences guide nucleases to RNA or DNA molecules with complementary sequences, resulting in degradation of the target nucleic acid. While recent work has illuminated many details of CRISPR-RNA-guided surveillance and target interference, the process of new sequence adaptation remains more mysterious. Initially, the goal of this research was to understand how new spacer sequences are acquired and integrated at CRISPR loci. High throughput sequencing of spacers acquired in in vivo adaptation assays revealed that some spacer sequences are reproducibly acquired in the I-F CRISPR system of Pseudomonas aeruginosa, and that the I-F CRISPR-guided surveillance complex enhances the efficiency of new spacer acquisition. We then used bioinformatic and in vitro acquisition assays to show that adaptation in many systems is dependent on the presence and phasing of sequence motifs in the transcriptional leaders of CRISPR loci. Collectively, these results expand our understanding of how CRISPR-Cas systems adapt to new threats. Following the emergence of SARS-CoV-2, and the ensuing international COVID-19 pandemic, my research goals pivoted to developing methods to track the spread of this coronavirus and to understanding how it was evolving. Long read genomic sequencing was used to determine the likely evolutionary origin of SARS-CoV-2 samples isolated from wastewater and human patients. This work led to the identification of isolates with large genomic deletions and shows that while these mutations cause a replication defect in the virus, similar mutations have appeared multiple times, independently in the evolution of SARS-CoV-2. Finally, we show that type III CRISPR-Cas systems can be repurposed for molecular detection of SARS-CoV-2 and investigate how these new diagnostic platforms can be improved.