PROJECT SUMMARY/ABSTRACT This project’s long-term goals are to understand the regulation of human mast cell (MC) function and to elucidate the roles of MCs in human health and disease. MCs are major effector cells in IgE-associated responses (e.g., asthma, allergy, parasite immunity). These cells also may contribute to certain autoimmune disorders and in the initiation of responses to pathogens and other agents. Upon activation, MCs undergo degranulation, leading to the secretion of many mediators (including stored products, e.g., histamine, heparin and proteases), as well as the production of lipid mediators, e.g., LTC4 and PGD2, and many cytokines and growth factors. Depending on the setting, MCs can have effector and/or immunoregulatory roles. Most research investigating MC development and function has employed various mouse models. However, we know considerably less about the mechanisms that regulate the activation of human MCs. Similarly, relatively little is known about how the perturbation of signaling pathways in human MCs can contribute to MC- associated pathology. This, in turn, has hampered the design of therapeutic agents for the treatment and/or prevention of allergies and other mast cell-associated diseases in human subjects. We recently developed a technology platform employing functional genomics, coupled with high-resolution single-cell confocal imaging, which can rapidly identify regulators of degranulation in human mast cells. We now propose to use this platform to identify key regulators of IgE/FceRI-dependent signal transduction pathways in cultured, donor- derived, primary human mast cells. Specifically, we will attempt to validate, in primary human mast cells, selected major degranulation regulators previously identified in rodent mast cells. We also will use targeted arrayed human CRISPR-Cas9 libraries to identify novel (i.e., previously unidentified) regulators of IgE/FceRI- dependent signaling pathways in primary human mast cells. In addition, we will investigate the functional significance of the KIT D816V mutation detected in mastocytosis patients by using the CRISPR-Cas9-based gene editing technologies to induce/correct the same mutation in human HMC-1 cell lines and primary human mast cells. To achieve our goals, we propose two aims. In Aim 1, we will identify novel human mast cell degranulation regulators and systematically define IgE/FceRI-dependent signaling pathways in primary human mast cells. In Aim 2, we will use CRISPR-Cas9 “prime editing” to perform mutational studies of HMC- 1 human mast cell lines and primary human mast cells. Together, this work will help create a detailed model of mast cell degranulation in human mast cells and will begin to analyze how the KIT D816V mutation may influence human mast cell biology.