| Summary: | Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2017. === Vita. Cataloged from PDF version of thesis. === Includes bibliographical references. === Targeted methods to perturb nucleic acid sequences or epigenetic modifications enable better understanding of nucleic acid function or dysfunction in disease. Particular CRISPR-associated (Cas) proteins are used for targeted DNA editing because they are easy to use, precise, and specific. CRISPR-Cas systems function in microbes as adaptive immune systems and are composed of endonuclease protein(s) with targeting guide RNAs that together provide interference against foreign nucleic acids. CRISPR-Cas systems exhibit wide diversity, with the ability to target DNA or RNA, and some large single effector Class 2 Cas proteins have been harnessed for genome engineering. There is an interest to find and characterize additional Class 2 Cas proteins, especially ones that target RNA, to enable further targeting capabilities. A large computational database search was conducted to find a large uncharacterized protein within 10 kilobases of a CRISPR array and from this search casl3b was identified. The casl3b loci may contain the genes csx27 or csx28 and one or two CRISPR arrays with canonical or long direct repeats. Experimentally, purified Casl3b processes its own CRISPR array(s) into crRNA(s) and Casl3b with either crRNA architecture targets single stranded RNA cleavage using its HEPN domains. SsRNA cleavage occurs at pyrimidine residues and is constrained by 5' and 3' protospacer-flanking sequences on the target. Casl3b also displays the collateral effect, a non-specific cleavage of ssRNAs after targeted cleavage. Casl3b with mutated HEPN domains lose ssRNA cleavage activity but maintain strong, targeted binding capacity. Genetically, Casl3b-mediated RNA interference occurs with Casl3b alone and is repressed by Csx27 or enhanced by Csx28. Casl3b is characterized as a Type VI-B CRISPR-Cas system and represents the second Class 2 Cas protein to target RNA, the other being Casl3a (C2c2). Future studies of Casl3b are warranted to better understand its functional mechanisms, specificity, role of small proteins, and acquisition. Casl3b could be developed into a suite of tools for transcriptome engineering to mediate RNA translation, splicing, or deposition of epitranscriptomic marks. Casl3b could also be utilized for RNA diagnostic or RNA imaging assays. Identifying and characterizing novel CRISPR-Cas systems opens new opportunities for utilizing Cas enzymes for biomedical advances. === by Neena Kenton Pyzocha. === Ph. D.
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