| Nature utilizes a number of methods for regulating gene expression via modulation of RNA stability. Factors involved in these processes include microRNAs, nucleotide modifications, structural elements, and ribonucleases, among others. In this dissertation, we aim to develop novel approaches for controlling gene expression through the manipulation of RNA stability. Prokaryotic CRISPR systems have provided a wealth of new tools for a range of molecular biology applications. A large proportion of these CRISPR-based technologies rely on CRISPR-associated nucleases to cleave either target DNA or RNA. The CRISPR endoribonuclease, Csy4 (Cas6f), is one such CRISPR protein, which specifically interacts with, and cleaves a target RNA hairpin. Here, we explore the use of Csy4 for destabilizing and stabilizing RNA transcripts in mammalian cells. We find that Csy4 is capable of knocking down gene expression when targeting the 5'UTR or coding sequence of a reporter mRNA. Additionally, Csy4 can rescue destabilized transcripts when targeted to the 3'UTR, and can promote their translation. We utilize the ability of Csy4 to stabilize target hairpin-containing cleavage products to induce RNA circularization of an engineered RNA splicing reporter. Further, we demonstrate the use of recombinant Adeno-Associated Virus (rAAV)-delivered circular RNA (circRNA) expression cassettes as platforms for gene expression in vivo. We find that these circRNAs can be translated to produce protein products in mice, and we observe expression differences between heart and highly proliferative liver tissue. The new approaches presented here provide starting points for the development of a number of new technologies including genetic 'safety switches' and genetic cassettes capable of therapeutic gene expression at lower rAAV vector dosages. |
