Aird, Eric2021-10-252021-10-252020-09https://hdl.handle.net/11299/225123University of Minnesota Ph.D. dissertation. September 2020. Major: Biochemistry, Molecular Bio, and Biophysics. Advisor: Wendy Gordon. 1 computer file (PDF); vi, 132 pages.Genome engineering, the ability to manipulate and modify genomes, has become a standard tool in life sciences and beyond. Programmable nucleases such as CRISPR-Cas9 have afforded the ability to target particular regions in genomes to make targeted changes. While genome editing technologies continue to flourish, the ability for CRISPR-Cas9 to deliver precise genomic modifications is in part hindered by the lower efficiencies of homology-directed repair (HDR). Additionally, delivery of these genome modifying reagents is hampered by current technological constraints. This dissertation describes our unique approaches to developing tools to improve both of these aspects of genome engineering. HUH endonucleases are a family of ssDNA binding proteins that bind sequence specifically to its target and have been co-opted for biotechnological purposes. We developed a Cas9-HUH fusion that increases precise editing outcomes on the order of 2- to 3-fold through covalent tethering of the HDR template. We also demonstrate application of this platform in concert with the development of a molecular tension sensor based on bioluminescent energy transfer (BRET). Next, we pivot to improving delivery of genome editors utilizing adeno-associated virus (AAV). We describe efforts to specifically target AAV to explicit cell types of interest via HUH-antibody selectivity. Building upon this method, we present a novel iteration of a next generation CRISPR-Cas9 based technology termed prime editor. We developed a split prime editor capable of being delivered by dual AAVs in vivo. Overall, the technologies and methodologies developed in this dissertation can readily be incorporated in various CRISPR-Cas workflows to enhance precise genome editing or specific targeting of various genome modifying reagents such as prime editor.enAAVBRETCRISPRHUH endonucleasePrimeThesis or Dissertation