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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 Enzymatic strategies for controlling and harnessing the oxidative power of O 2(Montana State University - Bozeman, College of Letters & Science, 2018) Machovina, Melodie M.; Chairperson, Graduate Committee: Jennifer DuBois; Robert J. Usselman and Jennifer L. DuBois were co-authors of the article, 'Monoxygenase substrates mimic flavin to catalyze cofactorless oxygenations' in the journal 'Journal of biological chemistry' which is contained within this dissertation.; Emerald S. Ellis, Thomas J. Carney, Fikile R. Brushett and Jennifer L. DuBois were co-authors of the article, 'Understanding how a cofactor-free protein environment lowers the barrier to O 2 reactivity' in the journal 'Journal of biological chemistry' which is contained within this dissertation.; Sam J. B. Mallinson, Rodrigo L. Silveira, Marc Garcia-Borras, Nathan Gallup were authors and Christopher W. Johnson, Mark D. Allen, Munir S. Skaf, Michael F. Crowley, Ellen L. Neidle, Kendall N. Houk, Gregg T. Beckham, Jennifer L. DuBois and John E. McGeehan were co-authors of the article, 'A promiscuous cytochrome P450 aromatic O-demethylase for lignin bioconversion' in the journal 'Nature Communications' which is contained within this dissertation.; Sam J.B. Mallinson was an author and Brandon C. Knott, Marc Garcia-Borras, Alexander W. Meyers, Lintao Bu, Japheth Gado, April Oliver, Graham P. Schmidt, J. Hinchen, Michael F. Crowley, Christopher W. Johnson, Ellen L. Neidle, Christina M. Payne, Gregg T. Beckham, Kendall N. Houk, John E. McGeehan and Jennifer L. DuBois were co-authors of the article, 'Enabling microbial syringol conversion through structure-guided protein engineering' submitted to the journal 'PNAS' which is contained within this dissertation.; Dissertation contains one article of which Melodie M. Machovina is not the main author.Dioxygen, one of Nature's most powerful oxidants, is essential for countless biological reactions. To harness this oxidant's power while minimizing toxicity, enzymes evolved to interact with O 2, activate it, and poise it for catalysis with substrates. This dissertation explores how two very different enzyme families, monooxygenases and a new class of cytochrome P450s, utilize this powerful oxidant. Previously, it was thought that cofactors are essential for O 2 activation; however, a subset of O 2-utilizing enzymes that catalyze direct reactions between substrate and O 2 was recently discovered, including nogalamycin monoxygenase (NMO). To probe how the protein environment affects thermodynamic and kinetic barriers of O 2 activation, we used a suite of techniques, including: UV/vis (transient and conventional) and electron paramagnetic resonance spectroscopies, O 2 consumption, high-performance liquid chromatography (HPLC), and cyclic voltammetry. Here, we provide evidence that the NMO mechanism has similar characteristics to that in flavoenzymes; in NMO, the substrate, acting in lieu of flavin, donates an electron to O 2, activating it to superoxide with the protein environment facilitating this by lowering the reorganization energy. The last half of this dissertation describes the discovery and engineering of a new class of cytochrome P450 enzymes that employ heme-iron oxygen activation to demethylate key lignin degradation products, forming central carbon intermediates that are precursors for bioplastics. The P450 GcoAB, comprised of the oxidase GcoA and the reductase GcoB, is efficient at demethylating G-lignin, but shows poor reactivity towards S-lignin. Using a structure-guided mutagenesis approach, we generated a variant, F169A GcoA, that is more efficient than wild-type at demethylating G-lignin and the only enzyme that efficiently degrades S-lignin. We characterized this variant, and the wildtype enzyme, using biochemical (UV/vis spectroscopy, HPLC), structural (X-ray crystallography), and computational (Molecular Dynamics and Density Functional Theory). Currently, we are testing the in vitro efficiency of additional variants evolved using a directed evolution approach. The results presented in the following chapters explore the mechanisms of several enzymes. Understanding how O2 is activated and utilized across diverse enzymatic systems provides valuable knowledge that can aid in future design and engineering of systems that use this 'green' oxidant, particularly for large-scale industrial applications.Item Developing and implementing genetic tools designed to understand host takeover by Chlamydia trachomatis.(Montana State University - Bozeman, College of Letters & Science, 2019) Kessy, Enock Joel; Chairperson, Graduate Committee: Blake WiedenheftChlamydia are gram negative obligate intracellular parasites that are responsible for millions of new infections in humans and animals every year. C. trachomatis is the number one cause of bacterial sexually transmitted infections in the United States, the number one cause of infectious blindness worldwide. Since 2001, there has been a steady increase in the number of new cases of C. trachomatis infections each year. Despite the prevalence and medical importance of C. trachomatis, we still know relatively little about the lifecycle of this parasite and the host factors that are essential for the lifecycle of C. trachomatis. To address this critical gap in our knowledge, my thesis work aimed to develop and implement genetic tools to understand host takeover by C. trachomatis. In this thesis I present results suggesting that I have transformed C. trachomatis with a plasmid carrying the Cas9 gene from Campylobacter jejuni. Additional experiments are necessary to determine if the CjCas9 is expressed, nuclease active, and functional for programable editing in C. trachomatis. In addition to my work aimed at developing a CRISPR-Cas9-based genetic engineering system in C. trachomatis, I also participated in a genome wide knockout screen aimed at identifying human genes necessary for completion of the C. trachomatis lifecycle. The CRISPR-Cas9 genome wide knockout screen identified 103 genes as critical factors for C. trachomatis. To validate results for the screen I have been involved in creating clonal cell lines with deletions in three of the genes that form the Adaptor Protein (AP) Complex (i.e., AP3S2, AP1B2 and AP1G2). The genes have been deleted and future experiments are aimed at measuring the impact of these genes on the C. trachomatis lifecycle.Item Modification of seed fatty acid composition by CRISPR/Cas9 targeting the fatty acid elongase1 in Camelina sativa(Montana State University - Bozeman, College of Agriculture, 2018) Ozseyhan, Mehmet Erkan; Chairperson, Graduate Committee: Chaofu LuThe low-input oilseed crop Camelina (Camelina sativa (L.) Crantz) is known for its high omega-3 (18:3) content, short growth season, and facile gene transformation. Camelina mostly contains unsaturated fatty acids, however its fatty acid composition needs optimization depending on the end uses, for example reduction of unsaturated fatty acid to use as biodiesels, or enhancing omega-3 fatty acid content to use as nutritional supplements. Very long chain fatty acid (VLCFAs, C20-C24), are undesirable for human consumption, and their accumulation in seed oil also needs to be diminished. VLCFAs are produced by the catalytic action of fatty acid elongase1 (FAE1), and Camelina contains three alleles of FAE1 genes (FAE1-A, FAE1-B, and FAE1-C) due to its allohexaploid nature. Recently, VLCFAs in camelina were decreased along with polyunsaturated fatty acids (PUFAs) using the RNA interference (RNAi) technology. A low VLCFA line was also isolated from ethyl methanesulfonate (EMS) induced mutants. Sequencing results indicated that FAE1-B gene was mutated and resulted in 60% reduction in VLCFAs, but other two FAE1 copies were presumably still active in the mutant. To address this multipleallele-knockout-at-once problem, here I investigated the effect of knocking out three alleles of FAE1 genes using CRISPR technology with egg cell-specific Cas9 expression. Due to the germline mutation, homozygous FAE1 knockout mutants were successfully created in a single generation. VLCFA accumulation was significantly decreased from 22% of total fatty acids in wild type to less than 2% in transgenic plants, and the C18 unsaturated fatty acids were improved since 18:1 substrates were diverted to desaturation pathway, rather than elongation. Analysis of the fatty acid composition of four transgenic generations indicated that the mutations that cause low VLCFA genotype were heritable. There was no significant difference observed in seed weight, plant height, total oil content, and seed germination in Cas9-induced mutants compared to the wild type. This study showed that polyploid Camelina can be modified rapidly and effectively through CRISPR/Cas9 to achieve desired fatty acid composition.Item Systems for studying the non-ubiquitous functions of the TATA-binding protein(Montana State University - Bozeman, College of Agriculture, 2003) Tucker, Tammy Alice; Chairperson, Graduate Committee: Edward E. Schmidt.Item Enzymatic recovery and in vitro culture of bovine primary oocytes(Montana State University - Bozeman, College of Agriculture, 1981) Connor, Henry CharlesItem Low dose tolerance vaccine platform, reovirus protein sigma 1 and treatment of autoimmunity(Montana State University - Bozeman, College of Agriculture, 2008) Rynda, Agnieszka; Chairperson, Graduate Committee: David Pascual.Effective treatments for multiple sclerosis (MS) are problematic due to its unknown etiology. Experimental autoimmune encephalomyelitis (EAE) in rodents mimics MS. Mucosal treatment of EAE with antigens to induce tolerance is effective, but requires large and/or multiple administrations, which introduces an allergy risk. We utilized reovirus adhesin, protein sigma 1 (p sigma1), to improve mucosal auto-antigen delivery and show that a single low-dose of p sigma1-based vaccines induces tolerance and prevents autoimmunity when administered nasally. We engineered three p sigma1-based vaccines carrying chicken ovalbumin (OVA-p sigma1) and/or myelin antigens (PLP:OVA-p sigma1, MOG-p sigma1). When mice were nasally immunized with OVA-p sigma1, tolerance to OVA was established. This tolerance resisted co-administration of mucosal adjuvants and peripheral challenge with OVA. P sigma1-mediated tolerance relied upon specific IL-10- producing regulatory T (T reg) cells, which inhibited OVA-specific CD4+ T cell proliferation. OVA-p sigma1 did not generate tolerance in IL-10-deficient mice presumably by a failure to induce T reg cells. Mucosal, but not systemic p sigma1 delivery, induced tolerance, while mice lacking mucosal inductive tissues were resistant to p sigma1-mediated tolerance. Likewise, PLP:OVA-p sigma1 and MOG-p sigma1 protected mice against relapsing-remitting or acute EAE, respectively. Protection against PLP139-151-induced EAE was accomplished by PLP:OVA-p sigma1, but not OVA-p sigma1, implicating antigen-specificity of p sigma1-mediated tolerance. Moreover, MOG-p sigma1, but not PLP:OVA-p sigma1, ameliorated MOG35-55-induced EAE via apoptosis of encephalitogenic CD4+ T cells. The PLP:OVA-p sigma1- or MOG-p sigma1-mediated protection against EAE depends on specific IL-10+ T reg cells and is supported by IL-4+ Th2-type cells. Adoptive transfer of PLP:OVA-p sigma1-primed T reg cells entirely prevented EAE development in mice; however, transfer of PLP:OVA-p sigma1-specific CD25- CD4+ Th2 cells significantly reduced and delayed clinical EAE. Aggressive EAE, due to the TGF-beta which induced activation of Th17 cells, was observed in mice dosed with PLP:OVA-p sigma1 and were functionally depleted of T reg cells. Concomitant inactivation of TGF-beta and T reg cells induced Th2 cells bias and re-established PLP:OVA-p sigma1-mediated protection against EAE. IL-10-producing B cells supported MOG-p sigma1-mediated protection against EAE, as MOG-p sigma1-dosed B cell-deficient mice developed attenuated disease. Adoptive transfer of T reg cells, but not Th2 or B cells from MOG-p sigma1-dosed B6 mice to diseased IL-10-/- mice, significantly accelerated recovery from EAE. These data demonstrate the feasibility of using p sigma1-based single-dose delivery system to prevent and/or treat autoimmunity.