PROJECT SUMMARY Natural killer (NK) cells are key effector lymphocytes in the innate immune system. They exhibit the remarkable abilities of detecting and eradicating transformed and virus-infected cells. NK cells also play a key role in the rejection of transplanted organs from allogeneic donors. Imbalances in NK cell-mediated killing are associated with major forms of human disease such as cancer, chronic infection, and immunodeficiency. To improve the efficacy of NK cell-based therapies, it is critical to identify the genes controlling the response of target cells to NK cell-mediated killing. In our preliminary studies, we performed genome-wide genetic screens to dissect target cell killing by an NK-like cell line. The screens isolated known regulators of NK cell killing but the majority of the hits were not previously linked to NK cell functions. The major goal of this pilot project is to validate and characterize these candidate genes using human primary NK cells. Each of these genes will be individually deleted in target cells using CRISPR genome editing. We will then determine how the knockout cells respond to killing by primary NK cells. Once a candidate gene is validated, we will further characterize its function and mechanism in NK cell-mediated cytotoxicity. Successful completion of this pilot project will substantially broaden our knowledge of how NK cells recognize and kill target cells. Insights gleaned from this work will help improve the efficacy and safety of immunotherapies for cancer and pathogen infection.