PROJECT SUMMARY
Natural killer (NK) cells are effector lymphocytes in the innate immune system. They destroy transformed
and pathogen-infected cells mainly through secreting the contents of membrane-enclosed cytolytic
granules, secretory lysosomes containing the pore-forming protein perforin and the serine proteases
granzymes. When a NK cell recognizes its target cell, the contact area between the NK cell and the
target cell forms a highly organized structure known as the immunological synapse. Cytolytic granules
then migrate toward the immunological synapse, where they fuse with the plasma membrane to release
their cytolytic molecules, a process known as cytolytic granule exocytosis. Once released from the NK
cell, cytolytic molecules enter the target cell and trigger cell death. Imbalances in cytolytic molecule
exocytosis cause major forms of human disorder such as immunodeficiency and chronic pathogen
infection. The molecular basis of cytolytic granule exocytosis in NK cells is still poorly understood. The
proposed research aims to bridge this knowledge gap. In our preliminary studies, we established an
assay to quantify cytolytic granule exocytosis in a physiologically relevant NK-like cell line. Furthermore,
we developed new platforms to genetically dissect complex exocytic pathways in mammalian cells using
unbiased CRISPR screens. In this research, we will take strategic advantage of these assays to dissect
cytolytic molecule exocytosis in NK cells using a genome-wide CRISPR screen. Next, we will validate
the candidate genes identified in the screen using a pooled secondary screen. Finally, we will validate
selected candidate genes using individual gene knockout. If successfully accomplished, this proposed
research will offer the first genome-scale view of the cytolytic exocytic pathway and will significantly
broaden our knowledge of NK cell functions. Insights acquired from this work will facilitate the
development of novel therapeutic strategies for immune diseases caused by defective cytolytic granule
exocytosis.