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Item Mechanisms of RNA-targeting CRISPR systems and their applications for RNA editing(Montana State University - Bozeman, College of Agriculture, 2022) Nichols, Joseph Edward; Chairperson, Graduate Committee: Blake Wiedenheft; This is a manuscript style paper that includes co-authored chapters.Genetic modification studies are central to understanding gene function and are the bedrock of molecular biology. The development of novel, CRISPR-based technologies for genome engineering in the last decade has revolutionized nearly every field of biology by simplifying the process of editing DNA genomes. In contrast, there are currently no comparable tools for editing RNA. Our goal is to develop facile CRISPR-based RNA editing methods that will transform our understanding of RNA metabolism, viruses and the repair pathways that govern RNA biology. I didn't initially come to MSU intending to study SARS-CoV-2, but the growing importance of this topic, combined with unanticipated intersections with my interest in CRISPRs, ultimately lead to several projects in this area. While participating in genomic surveillance, we identified a naturally occurring deletion within ORF7a, a viral accessory protein. We determined that this deletion results in the loss of function of ORF7a, limiting the virus' ability to evade host interferon responses, and reduced viral fitness. My focus then moved to Type-III CRISPR systems. While CRISPR has become synonymous with genome engineering, these systems naturally evolved in prokaryotes as an adaptive immune system against bacteriophages. Type-III CRISPR systems are unique, as they are one of two groups of CRISPR systems to target RNA rather than DNA. To develop type III systems for editing RNA, we designed and purified a series of type III complexes and showed that these systems function as programable nucleases. We then adapted a method for targeted RNA repair in vitro following cleavage and demonstrate that this approach results in edited RNA. In addition to cleaving the RNA target, target recognition by type III CRISPR systems also activates a polymerase domain that generates signaling molecules that activate ancillary CRISPR nucleases. Working with several members of the team, I set out to determine substrate preferences for each ancillary nuclease in Thermus thermophilus. We expected that activating these immune components would result in dramatic changes in bacterial growth kinetics. However, my experiments failed to identify a reliable phenotype, suggesting that this expression system is not a faithful representation of Type-III immunity.Item 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.Item Narratives and the Policy Process: Applications of the Narrative Policy Framework. Chapter 5: Lost in Translation: Narrative Salience of Fear > Hope in Prevention of COVID-19(Pressbooks, 2022) Peterson, Holly L.; Zanocco, Chad; Smith-Walter, AaronUsing short, policy-image-like narratives, we explore the relationship between narrative agreement and narrative impacts in the case of COVID-19 in the US. Building upon previous research which identified attention narratives focusing on problems “stories of fear” and those focusing on solutions “stories of hope,” we use a narrative survey experiment of the general public (n=1000) to test the salience of problem and solution narratives and if they impact agreement with Center for Disease Control (CDC) prevention guidelines. Our findings are 1) fear story agreement is partisan but hope story agreement is not 2) fear story is the more salient of the two, 3) narrative agreement for both fear and hope were related to CDC safety guideline agreement, but were partisan, and 4) exposure to neither narrative impacted likelihood to agree with the guidelines as compared to a control group. Our findings are consistent with previous work indicating a Democratic party preference for stories of fear, where Democrats were more likely to support policy action. While we find that agreement with our narratives and guidelines is related, neither narrative treatment successfully altered support for CDC guidelines, suggesting a potential limit for the influence of narratives to either change or reorder existing preferences in highly salient and partisan issue areas like COVID-19 and suggesting a need for more research into the dynamics of narrative attention.Item Aerosol stability of severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2)(Montana State University - Bozeman, College of Agriculture, 2022) Bushmaker, Trenton John; Chairperson, Graduate Committee: Raina K. Plowright and Vincent J. Munster (co-chair); Neeltje van Doremalen and Dylan H. Morris were authors and Myndi G. Holbrook, Amandine Gamble, Brandi N. Williamson, Azaibi Tamin, Jennifer L. Harcourt, Natalie J. Thornburg, Susan I. Gerber, James O. Lloyd-Smith, Emmie de Wit and Vincent J. Munster were co-authors of the article, 'Aerosol and surface stability of SARS-CoV-2 as compared with SARS-CoV-1' in the journal 'New England journal of medicine' which is contained within this thesis.; Claude Kwe Yinda and Dylan H. Morris were authors and Myndi G. Holbrook, Amandine Gamble, Danielle Adney, Cara Bushmaker, Neeltje van Doremalen, Raina K. Plowright, James O. Lloyd-Smith and Vincent J. Munster were co-authors of the article, 'Comparative aerosol stability of SARS-CoV-2 variants of concern' submitted to the journal 'Emerging infectious diseases - CDC' which is contained within this thesis.The routes of transmission of the zoonotic pathogen severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) have been extensively studied to understand the spread at individual and population levels. Aerosol particles produced by infected individuals and the deposition patterns inhaled are known to affect the virulence of bioaerosol pathogens. Droplet nuclei particles (< 5 microns) aerosols typically deposit within the alveolar spaces of the lungs, whereas droplet (>5 microns) aerosols typically deposit within the nasopharyngeal and tracheobronchial regions of the respiratory tract. A few studies have evaluated pulmonary disease following droplet nuclei size particles of SARS-CoV-2 aerosol inhalation in African green monkeys and golden hamsters, concluding that both models have mild respiratory disease representative of human disease. More importantly, human participants with SARS-CoV-2 infections have been studied to look at the generation of particles during breathing, talking, and singing; the study concluded droplet nuclei particles accounted for 85% of the copies of virus produced and play a significant role in transmission. However, the environmental persistence of the aerosolized droplet nuclei particles, and the likely role of environmental persistence in driving transmission, is unknown for SARS-CoV-2. In these studies, we show the changing aerosol stability of SARS-CoV-2 during the supplanting waves of Variants of Concern (VOC). With the determination of viable viral particles characterized over time, we can make inferences about the role VOC and aerosol transmission have in driving population-level pathogen transmission. A secondary objective of these studies was to characterize the role those evolving mutations have had on viral entry and aerosol durability. Our work suggests that aerosol stability may be important in driving some population-level phenomena (e.g., indoor transmission, including superspreader events) but given the short infected-to-naive transmission transit time, the variation in the duration of aerosol stability among VOCs may not explain the difference in transmission rates of VOCs. This data will be useful for assessing the future evolution of aerosol transmission of SARS-CoV-2.