Programming Bacterial Gene Expression Using Synthetic CRISPR-Cas Transcriptional Regulators

Thesis (Ph.D.)--University of Washington, 2019Bacteria play a central role in biosynthesis to produce value-added organic chemicals due to its diverse carbon and energy source preferences. Implementing synthetic transcriptional regulation devices can advance our ability to modify gene expression in bacteria for engineering production strains. The CRISPR-Cas activation (CRISPRa) system, a programmable transcriptional activator with wide applications in eukaryotic organisms, has been under-utilized in bacterial due to the lack of efficient transcriptional activation domains. This work describes our contribution to the development and understanding of bacterial CRISPR-Cas-based transcriptional regulation devices. We screened novel bacterial activation domains to be used as CRISPR-Cas activators in E. coli, and optimized our strongest activation domain, SoxS into a programmable CRISPR activator. In addition, we investigated the properties of the well-established CRISPRi repression system and found that partial repression can be achieved by controlling the expression level of the sgRNA. Combining CRISPRa and CRISPRi, we demonstrated inducible simultaneous up- and down-regulation of a dual reporter gene and the CRISPR-mediated regulation of the ethanol bioproduction pathway. Moreover, we also learned important properties of the bacterial CRISPRa system which is much more restrictive than the eukaryotic CRISPRa system. CRISPRa activity is dependent on the sigma factor that the promoter recruits, the baseline strength of the promoter, the sequence composition of the promoter, the presence of nearby transcription factors, and the precise positioning of the scRNA target. We attempted to relieve these restrictions by implementing an engineered Cas protein that can bind to a wider range of targets. Lastly, we describe our efforts to transport the CRISPRa system into other non-E.coli bacteria. CRISPR-SoxS activator proved to be active in Pseudomonas putida, but not in the other organisms we tested. Therefore, we propose to characterize host-specific activation domains for bacteria whose cellular machineries are incompatible with SoxS. Together, this work provided a novel programmable gene activation device in bacteria and sets up the foundation for the development of complex, broad-host-range bacterial cellular devices for biosynthetic applications