CRISPR-Cas technologies enable robust manipulation of genetic material, and have been instrumental in advancing a wide range of fields across the life sciences. Specifically, with the ability to correct or alter faulty genes, genome editing tools promise to transform the field of genetic medicine. Current CRISPR-based editors [nucleases, base editors (BEs), and prime editors (PE)] can be programed to induce efficient mutagenesis/repair, conversion, and polymerization, respectively. Presently, nucleases - the most clinically advanced genome editors - suffer from inadequate control of genome editing outcomes. Over time, the field has focused on precision editors such as BEs and PEs that do not rely on double-strand breaks and greatly improve the safety and control of genome editing outcomes.

Despite these advances, challenges such as targeting scope, accuracy and in vivo delivery represent major hurdles for the therapeutic application of next-generation editing systems such as BE and PE. In this thesis, I focus on alleviating some of the key obstacles associated with effective genome editing by improving the unique properties of a compact Cas9 orthologue (Nme2Cas9 from Neisseria meningitidis). The bulk of my thesis consists of protein engineering efforts to improve the activity and targeting scope of Nme2Cas9-derived editing systems. My later work focuses on the development of chemically stabilized guide RNAs, providing a path to facilitate in vivo delivery in a variety of formats. Overall, the advances presented in this thesis contribute to the versatility of CRISPR-based genome editing systems for a variety of therapeutic and research applications.