Chappell, James2022-12-212022-122022-11-08December 2Villegas Kcam, Maria Claudia. "Development of trans-acting RNA guided tools to control gene expression in bacteria." (2022) Diss., Rice University. <a href="https://hdl.handle.net/1911/114170">https://hdl.handle.net/1911/114170</a>.https://hdl.handle.net/1911/114170EMBARGO NOTE: This item is embargoed until 2024-12-01The ability to control gene expression is a transformative capability that allows scientists to uncover gene function or induce valuable phenotypes and cell fates important for applications in biotechnology and biomedicine. We are specifically interested in the development of trans-acting regulators that can target non-synthetic sequences allowing for applications in endogenous pathways and high throughput screenings. CRISPR-Cas editors and regulators have provided an excellent toolbox for this purpose. However, current bacterial CRISPR activators (CRISPRa) present several challenges such as the lack of a global activation domain (AD) that can be used across bacteria and the restricted availability of functional binding sites in non-synthetic targets. To address these limitations we generated a highly modular CRISPRa platform where each element of the system (dCas9, sgRNA, AD, and target gene) can be easily modified, and thus we could screen for different CRISPRa designs. Initially, we identified a new AD for CRISPRa that we harnessed to explore different AD recruitment strategies and create the modular CRISPRa platform. Next, we evaluated the use of circularly permuted variants of the dCas9 protein to identify variants capable of activating gene expression from targeting positions previously identified as non-functional sites for CRISPRa. Additionally, we used the modular platform to create the first Type I-E CRISPRa system for bacteria and uncovered how its distinct properties differ from the previously used Type II- CRISPRa. With this study, we open the possibility for new CRISPRa systems harnessing different CRISPR systems to obtain distinct properties. Finally, we applied the CRISPRa system in Streptomyces venezuelae, a bacteria of interest for the production of new antibiotics. We characterized the CRISPRa system in S. venezuelae and targeted an endogenous pathway to induce the production of an antibiotic product. We demonstrate that the modular CRISPRa platform can be adapted to provide a more flexible and versatile tool that will expand our ability to activate and control bacterial genomes.application/pdfengCopyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.CRISPRagene activationtranscriptionRNAgene expressiontranslational fusionRibozymechimeric proteinThesis2022-12-21