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Structural characterization of the Csa3/cA4 complex - a nexus for class 1 CRISPR-Cas immune response coordination & establishing a cure for highly efficient galectin expression
(Montana State University - Bozeman, College of Letters & Science, 2024) Charbonneau, Alexander Anthony; Chairperson, Graduate Committee: C. Martin Lawrence; This is a manuscript style paper that includes co-authored chapters.
Though Class I CRISPR-Cas systems, primarily Type I and Type III, are the most abundant CRISPR systems in archaea and bacteria, mechanisms driving their immune response regulation are not well understood. Csa3 family transcription factors, composed of N-terminal CARF and C-terminal winged helix-turn-helix domains, are frequently encoded within Type I CRISPR-Cas systems. Csa3 transcription factors are hypothesized to bind cyclic oligoadenylate (cOA) second messengers produced by Type III interference complexes, likely modulating their DNA-binding activity. Therefore, we investigated the interaction between Csa3a and cyclic tetra-adenylate (cA4). Isothermal titration microcalorimetry showed S. solfataricus Csa3a binds cA4 at biologically relevant concentrations in an entropically driven interaction. Ring nuclease assays revealed Csa3a lacks self-regulatory phosphodiesterase activity exhibited by other CARF domain proteins. We crystallized and solved the structure of the Csa3/cA4 complex, which revealed conserved motifs are responsible for cA4 binding and illuminated significant conformational changes induced by the interaction. We also identified an 18-bp palindromic motif, which we designated CAPPa, that is conserved in the 27 sequenced members of the order Sulfolobales, and shows synteny with Csa3a and acquisition genes in these genomes. We found Csa3a binds CAPPa in a nonspecific, cooperative, and cA4-independent manner. These characteristics suggest a more complex method of transcriptional regulation than previously hypothesized. However, the interaction between Csa3a and cA4 confirmed here signifies a nexus between Type I and Type III systems; we thus propose a model in which this interaction coordinates the two arms of an integrated immune system to mount a synergistic, highly orchestrated, adaptive immune response. We applied the workflow designed to produce significant protein quantities for crystallographic studies of Csa3a to the study of Homo sapiens galectin proteins, a family of beta-galactoside-binding proteins. Here, we identified a putative autoinhibitory mechanism affecting traditional IPTG-induction methods by characterizing IPTG-binding capabilities of galectins and quantifying basal protein expression over various IPTG concentrations. To bypass this predicted feedback loop, we employed a highly efficient and approachable autoinduction method, resulting in a 7-fold increase in protein expression. Much of this work was done in the context of a course-based undergraduate research experience with great success.
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.
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.
Allosteric activation of the CRISPR-associated transcription factor Csa 3 by cyclic tetra-adenylate (cA 4)
(Montana State University - Bozeman, College of Letters & Science, 2020) Charbonneau, Alexander Anthony; Chairperson, Graduate Committee: C. Martin Lawrence
The CRISPR-Cas immune system provides adaptive and heritable immunity to archaea and bacteria to combat viral infection, and is a source of biochemical tools to researchers. This work combines structural biology and biochemical approaches to provide insight into mechanisms prokaryotes use to control the CRISPR-Cas immune system, linking subsystems into a coordinated response. The first structure of S. solfataricus Csa3 determined by Lintner et al. revealed a dimer with a C-terminal wHTH DNA-binding domain and an N-terminal CARF domain with a putative ligand binding site predicted to bind a two-fold symmetric molecule with both negatively charged and hydrophobic/aromatic moieties, such as dinucleoside polyphosphates or nucleic acid molecules. 1 Later, analysis by Topuzlu et al. of the A. fulgidis Csx3 structure containing a 4 base RNA molecule in a binding pocket revealed similarities between Csx3 and the Csa3 CARF domain, and suggested CARF proteins could bind cyclic or pseudosymmetric linear RNA tetranucleotides represented by the ring-shaped RNA density in the Csx3 binding pocket. 2 Functional studies with S. islandicus Csa3 identified that SiCsa3 regulates transcription of acquisition genes (cas1, cas2, and cas4) and several CRISPR loci. 3,4 Additionally, two groups simultaneously showed that the Type III surveillance complexes produce cyclic oligoadenylate messengers, including cyclic tetra-adenylate (cA 4), which allosterically regulate the RNase activity of Csm6 and Csx1, other CARF proteins. 5,6 These advances support the original predictions by Lintner et al. and suggest that Csa3 binds a cyclic RNA as proposed by Topuzlu et al. Binding of cA 4 likely allosterically causes conformation changes in the wHTH, and regulates the protein's transcriptional regulation. We present the crystal structure of S. solfataricus Csa3 complexed with cyclic tetraadenylate (cA 4). cA 4, as predicted, 1 is bound in the CARF domain 2-fold symmetric pocket, which stimulates conformational changes in the C-terminal domain. Additionally, we identify the presence of a palindromic predicted binding motif upstream of the Type I-A(2) acquisition cassette and CRISPR loci C and D, and reveal through EMSA analysis that Csa3 binds dsDNA nonspecifically with high affinity. Finally, ring nuclease activity is not detected in Csa3, suggesting longer term potentiation of the cA 4 in Csa3 than observed for Csx1/Csm6. 5,6
Mechanisms of CRISPR-mediated immunity in Escherichia coli
(Montana State University - Bozeman, College of Letters & Science, 2019) van Erp, Paul Bertram Geert; Chairperson, Graduate Committee: Blake Wiedenheft; Gary Bloomer, Royce Wilkinson and Blake Wiedenheft were co-authors of the article, 'The history and market impact of CRISPR RNA-guided nucleases' in the journal 'Current opinion in virology' which is contained within this thesis.; Ryan N. Jackson and Joshua Carter were authors and Sarah M. Golden, Scott Bailey and Blake Wiedenheft were co-authors of the article, 'Mechanism of CRISPR-RNA guided recognition of DNA targets in Escherichia coli' in the journal 'Nucleic acids research' which is contained within this thesis.; Angela Patterson was an author and Ravi Kant, Luke Berry, Sarah M. Golden, Brittney L. Forsman, Joshua Carter, Ryan N. Jackson, Brian Bothner, and Blake Wiedenheft were co-authors of the article, 'Conformational dynamics of DNA binding and CAS3 recruitment by the CRISPR RNA-guided cascade complex' in the journal 'ACS chemical biology' which is contained within this thesis.; Tanner Wiegand, Royce A. Wilkinson, Laina Hall, Dominick Faith and Blake Wiedenheft were co-authors of the article, 'Protein overexpression reduces specific phage infectivity in prokaryotic argonaute screen' which is contained within this thesis.; Dissertation contains three articles of which Paul Bertram Geert van Erp is not the main author.
Prokaryotes are under constant threat from foreign genetic elements such as viruses and plasmids. To defend themselves against these genetic invaders prokaryotes have evolved extensive defense mechanisms. In this thesis I explore two such defense systems: prokaryotic Argonautes and CRISPR-systems. CRISPR-systems acquire short sequences derived from foreign genetic elements and store them in the CRISPR locus. In subsequent rounds of infection these stored sequences are used as guides by Cas proteins to target the invaders. Escherichia coli K-12 contains a type I-E CRISPR system, consisting of two CRISPR loci and eight cas genes. five of these cas genes, together with and 61-nucleotide CRISPR-RNA guide form the RNA-guided surveillance complex Cascade. This complex finds and binds foreign DNA targets that are complementary to its RNA guide. After target binding the helicase/nuclease Cas3 is recruited to the Cascade-DNA complex for destruction of the target. The goal of this research is to understand the molecular mechanisms that lead to target recognition and destruction in the type I-E CRISPR systems. Atomic resolution structures of the proteins involved in these CRISPR systems provide the blueprints of these proteins machines. Structure guided mutational analysis coupled with in vivo and in vitro biochemical experiments are used to investigate the underlying molecular mechanisms of this CRISPR system. Together, these results explain the rules of target recognition and Cas3 recruitment. Prokaryotic Argonautes have been hypothesized to defend against mobile genetic elements such as plasmids and viruses through guided nuclease activity. To test this hypothesis, we overexpressed 8 phylogenetically diverse prokaryotic Argonautes proteins in Escherichia coli and challenged them with seven bacteriophages. This resulted in robust protection against phage Lambda and phage P1 by four of the tested Argonautes, while little impact on phage infectivity was observed for the other phages tested. However, control experiments with a nuclease inactive Argonaute mutant and expression of an unrelated control protein showed similar protection against phage Lambda and phage P1. Collectively, our data suggest that protein overexpression in general, rather than Argonaute expression in particular, results in protection against 2 specific phages.
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 Wiedenheft
Chlamydia 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.
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 Lu
The 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.
Expression and purification of two CRISPR-CAS proteins, Csm3 and Csm5 from Mycobacterium tuberculosis
(Montana State University - Bozeman, College of Letters & Science, 2015) Hashimi, Marziah; Chairperson, Graduate Committee: C. Martin Lawrence
One third of the World's population is infected with tuberculosis (TB). TB disease is caused by bacterium called Mycobacterim tuberculosis, which is a facultative intracellular parasite that is transferred through the air from one person to another in close contact. A six month course of four antimicrobial drugs is the only current treatment for drug-sensitive TB. However, multi-drug resistance TB is difficult to treat. Phage therapy might be one answer as a treatment for multi-drug resistance TB. In order for phage therapy to have a chance against TB, the immune system of bacteria, known as CRISPR/Cas needs to be inhibited. Our lab has taken a structural and biochemical approach to try to understand the CRISPR/Cas system in M. tuberculosis. We have cloned, expressed, and purified individual Csm proteins from the H37Rv M. tuberculosis strain. Two Csm protein, Csm3 and Csm5 were successfully purified to homogeneity in yields suitable for structure and biochemical studies. While to date, each has failed to produce crystals, the ability to the express and purify each of these proteins will allow further biochemical characterization of Csm3 and Csm5.
From immunology to MRI data anlysis : problems in mathematical biology
(Montana State University - Bozeman, College of Letters & Science, 2015) Waters, Ryan Samuel; Chairperson, Graduate Committee: Tomas Gedeon
This thesis represents a collection of four distinct biological projects rising from immunology and metabolomics that required unique and creative mathematical approaches. One project focuses on understanding the role IL-2 plays in immune response regulation and exploring how these effects can be altered. We developed several dynamic models of the receptor signaling network which we analyze analytically and numerically. In a second project focused also on MS, we sought to create a system for grading magnetic resonance images (MRI) with good correlation with disability. The goal is for these MRI scores to provide a better standard for large-scale clinical drug trials, which limits the bias associated with differences in available MRI technology and general grader/participant variability. The third project involves the study of the CRISPR adaptive immune system in bacteria. Bacterial cells recognize and acquire snippets of exogenous genetic material, which they incorporate into their DNA. In this project we explore the optimal design for the CRISPR system given a viral distribution to maximize its probability of survival. The final project involves the study of the benefits for colocalization of coupled enzymes in metabolic pathways. The hypothesized kinetic advantage, known as 'channeling', of putting coupled enzymes closer together has been used as justification for the colocalization of coupled enzymes in biological systems. We developed and analyzed a simple partial differential equation of the diffusion of the intermediate substrate between coupled enzymes to explore the phenomena of channeling. The four projects of my thesis represent very distinct biological problems that required a variety of techniques from diverse areas of mathematics ranging from dynamical modeling to statistics, Fourier series and calculus of variations. In each case, quantitative techniques were used to address biological questions from a mathematical perspective ultimately providing insight back to the biological problems which motivated them.
Crenarchaeal virus-host systems : structure-function studies of crenarchaeal viruses and prokaryotic adaptive immunity
(Montana State University - Bozeman, College of Letters & Science, 2011) Lintner, Nathanael Greenleaf; Chairperson, Graduate Committee: C. Martin Lawrence; Valerie Copie (co-chair)
Virus-host interactions are one of the most important drivers of microbial ecology and evolution. Among the three domains of life, the least is known about viruses infecting members of the domain Archaea. This work combines a traditional hypothesis-driven molecular and biochemical approach with a structural genomics-like approach of pursuing a large number of structural targets to gain a detailed molecular understanding of a model Crenarchaeal virus-host system, Sulfolobus turreted icosahedral virus (STIV) and its Sulfolobus host. This work specifically focuses on the viral protein, A81, a putative transcriptional regulator associated with the prokaryotic adaptive immune system, CRISPR-Cas, and a large protein-RNA complex involved in CRISPR-mediated DNA interference. A81 is a protein-of-unknown-function encoded by the STIV genome. The structure of STIV-A81 reveals a unique ring-shaped octameric assembly. While structural homology-based searches fail to reveal a function for A81, the central pore has a size and charge consistent with a potential interaction with single-stranded nucleic acid. The structure of the CRIPSR-associated protein, Csa3 reveals a putative 2-domain transcription factor. The N-terminal domain is a variation on the di-nucleotide binding-domain that orchestrates dimer formation. There is a conserved 2-fold symmetric pocket on the dimer axis that likely represents a regulatory ligand-binding site implying a small-molecule regulator of CRISPR/Cas. The C-terminal domain is a winged helix-turn-helix common among transcription factors. The domain architecture of Csa3 suggests a small molecule regulator of CRISPR/Cas in the Crenarchaea. The CRISPR-associated complex for antiviral defense (CASCADE) is predicted to be central to CRISPR-mediated DNA-interference in many bacteria and Archaea. We isolated components of an archaeal CASCADE from Sulfolobus solfataricus. Csa2 expressed in S. solfataricus co-purifies with Cas5a, Cas6, Csa5 and Cas6-processed CRISPR-RNA (crRNA). Csa2, the dominant protein, forms a stable complex with Cas5a. A recombinant Csa2-Cas5a-complex is sufficient to bind crRNA and complementary ssDNA. Transmission electron microscopy reveals an extended helical complex of variable length, perhaps due to substochiometric amounts of capping factors. Csa2 displays a crescent-shaped fold including a modified RNA-recognition motif (RRM) plus two additional domains present as insertions into the RRM. A preliminary model for this and other CASCADEs is proposed.

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