156-OR: Genome-Wide CRISPR Screen Identified That Alternative Splicing Regulates in Insulin Secretion

Pancreatic β cell functionality, especially glucose-stimulated insulin secretion (GSIS), is pivotal in diabetes pathophysiology. The impairment of GSIS is not only evident in Type 2 diabetes (T2D) but also during the early stages of Type 1 diabetes (T1D). In addition, enhancing GSIS also holds significant promise for improving the functionality of stem cell-derived β cells (SC- β cells). Addressing this limitation offers new opportunities in cell-based diabetes therapy. To uncover the intricate regulatory mechanisms of insulin secretion, we developed a unique reporter cell line carrying NPY-pHluorin transgene to visualize insulin exocytosis at single-cell level and conducted a genome-wide CRISPR screen using flow cytometry system in NIT-1 cells. Notably, our study identifies alternative splicing (AS) as a pivotal factor in insulin secretion regulation. Employing genetic Sf3b1 knockout - a key component in AS machinery in NIT-1 cells, results in a surprising outcome: increased insulin secretion and improved GSIS. Consistently, using chemical inhibitors such as FR901464 to suppress AS activities, we observed the same phenotype in β cell lines and pancreatic islets. To unravel the underlying mechanisms, we performed comprehensive RNA-seq and alternative splicing-seq analyses in Sf3b1 knockout cell lines and cells treated with AS inhibitor, revealing alterations in genes related to the insulin production machinery and synaptic/exocytic vesicle traffic pathways. Our findings emphasize the significant role of AS in modulating β cell function. This research bridges critical gaps in our current understanding and sets the stage for innovative approaches to address the intricate challenges posed by diabetes.