PROJECT SUMMARY Defining the mechanisms that detect, signal, and repair distinct forms of DNA damage in human cells has been a major research focus for decades. At this point, we know a lot about how different DNA lesions are recognized, how repair processes are regulated, and how defects in those processes alter susceptibility to human diseases, including cancer and neurological disorders. Progress towards comprehensive understanding, however, has been stalled by two main challenges: we lack technologies for robustly detecting the activity of many individual repair processes in cells, and even when we can isolate specific forms of repair, redundancies and complexities in pathway-level organization obscure or conflate the roles of component factors. Our lab has developed and applied innovative functional genomics approaches to interrogate systems of DNA damage response with unprecedented resolution, and as a result, we have identified novel relationships between DNA repair genes. In parallel, we have elucidated cellular determinants of genome-editing tools, with and without collaborators. A key inference from one of our genome-editing studies was that prime editing tools, which use reverse transcription to ‘write’ programmed DNA sequence changes into otherwise intact genomes, often inadvertently model substrates of DNA mismatch repair (MMR) at targeted genomic loci. This proposal expands on that observation. In one research trajectory, we propose to resolve genetic pathways of MMR in human cells by pairing prime editing with functional genomics approaches. Results from this trajectory will determine how programmed DNA mispairs of various types stimulate different downstream MMR processes, refine our understanding of DNA strand selection in MMR, and elucidate genomic features that impact MMR. In a second research trajectory, we propose two technology development efforts, one to identify sequence features that allow prime edits to evade MMR detection, and the other to