Project Summary DNA is the blueprint for life. It encodes the instructions for building and maintaining all organisms, from prokaryotes to humans. Despite this, DNA is frequently damaged. In fact, estimates suggest that each human cell may experience as many as ~105 lesions per day. To protect their genomes, organisms have therefore evolved a sophisticated set of mechanisms that sense, signal, and repair chemically diverse forms of DNA damage and, when damage cannot be repaired, induce programs of cell death. Decades of work have provided an extensive ?parts list? of these mechanisms in human cells. However, a major challenge to our understanding remains: We do not know how these mechanisms work together at the systems level to ensure response flexibility across conditions or enable compensation when one mechanism fails. This lack of systematic knowledge is problematic. It limits our comprehension of human diseases, such as cancers with DNA repair deficiencies, and it challenges our ability to develop and improve medical therapies that exploit response activities. We have recently developed functional genomics approaches that enable systematic interrogation of gene function in human cells, and with these tools, we propose to address this gap in knowledge. The fundamental logic behind our approaches is simple. We pair CRISPR-based genetic perturbation techniques with scalable methods for obtaining high-content phenotypes, such as single-cell RNA-sequencing. This allows us to collect data rich readouts of cell behavior across cells in which we have perturbed the function of many genes. With such data, we can infer functional relationships between genes and delineate genetic pathways. Here, I propose to use two of these technologies to map DNA damage response mechanisms in human cells, with the goals of improving genome editing technologies (Project 1) and achieving deeper understanding of drug responses during cancer therapy (Project 2). To enable the first project, we demonstrate a new approach that pairs CRISPR-based genetic screens with deep sequencing of DNA repair junctions to generate high-content readouts of DNA repair. We establish this approach using Cas9 from Streptococcus pyogenes and propose work that will serve as a roadmap for understanding genome editing technologies in the future.