PROJECT SUMMARY The ability to install precise genetic changes is a longstanding goal in biology. While there are over 6,000 known disorders with monogenic origins, estimates are that only 10% of these are currently treatable. Most genetic variants associated with disease are single point mutations which are potentially correctable via systems that exchange a single DNA base (base editors). Base editors treat point mutations by appending a deaminase enzyme that catalyzes single nucleotide changes to a programmable DNA binding protein (Cas9) that localizes the editor to its target. This technology has seen overwhelming success with adenine base editors (ABEs) entering the clinic within just five years of their initial report. Notably, ABEs do not generate double-stranded breaks, making them an ideal candidate for genome editing—especially in stem cells which suffer low genome editing efficiency and large rearrangements or deletions in DNA in response to DNA cleavage. The correction of point mutations in the genomes of stem cells has the potential to provide essential cell-based therapies for immunodeficiency and neurodegenerative diseases. However, the capacity of this approach is limited by editing promiscuity at neighboring bases. This constrains target selection to a narrow range of mutations where nearby off-target edits would not negate the effect of the edit or induce additional pathogenic mutations. For instance, mutations causing severe combined immunodeficiency (SCID) and Hurler syndrome are potentially reversible by adenine targeting base editors, yet inaccessible by current methods due to the proximity of another editable adenine bases to the target site. Therefore, there is an unmet clinical need for the advent of precision editors capable of precisely targeting mutations in hematopoietic stem and progenitor cells (HSPCs) to produce edited cells for autologous transplantation. My proposal describes a two-pronged approach to developing precision base editors that both extends existing technology and creates an entirely new kind of editing enzyme with intrinsic properties that prevent off-target edits. Our approach provides key insights into genome editing mechanisms that can be harnessed for treatment of a wide range of diseases.