Project Summary/Abstract Alternative splicing (AS) is the fundamental mechanism of generating isoform diversity in eukaryotic cells. AS occurs at an especially high frequency in the retina and other nervous system tissues, contributing to many important cellular and physiological functions within the cells, including tightly regulated and complex processes like neuronal development. Moreover, dysregulation of AS can have a substantial impact on retinal survival and function. Retinitis pigmentosa (RP) is a group of inherited retinal diseases that cause dysfunction and degeneration of the light-sensitive photoreceptor layer of the eye, resulting in irreversible vision loss and blindness in over 1.5 million people worldwide. Yet, therapeutic options for these patients remain limited. Many mutations that cause mis-splicing of a gene cause RP. Furthermore, defects in the regulation of AS, including mutations in spliceosome components and other associated splicing factors, also cause disease. It is unknown why retinal cells are so particularly, and often exclusively, susceptible to aberrant splicing, despite these defects often occurring in either ubiquitously expressed genes or from mutated splicing factors found in all tissues of the body. By combining novel long-read RNA-sequencing technology with CRISPR engineering and 3D human stem cell organoid models, we can acquire a detailed understanding of AS and learn how dysregulated AS contributes to retina-specific disease. To further investigate the AS landscape in human retinal development and disease, this proposal aims to 1) comprehensively examine the AS events in rod and cone photoreceptors that occur during differentiation of human stem cell-derived retinal organoids, 2) investigate how splicing factor mutations alter normal AS in the retina compared to other neurons, and 3) understand how dysregulated gene expression that results from aberrant splicing causes retina-specific degeneration. During the mentored phase of this proposal, I will take advantage of the many strengths of my multidisciplinary team of mentor/co-mentors, advisors, and collaborators to perform and analyze the single cell long-read transcriptomic experiments with the developing retinal organoids and acquire the training needed regarding brain organoid differentiation and CRISPR methodologies to successfully transition myself to the independent research phase. In the independent phase, I will use the splicing factor mutant cell lines generated in the Zack lab to further understand the mechanism(s) of retinal-specific degeneration caused by dysregulated AS events. The experiments proposed will not only provide the retinal field with a more complete understanding of AS in the retina, knowledge which can possibly be harnessed to design new treatment options for RP, but the training plan we have developed will also provide a robust pathway to establishing my successful and productive independent research career that extends well be...