PROJECT ABSTRACT Hearing loss and balance disorders are the two most prevalent disabilities. Over 6% of people worldwide suffer from disabling hearing loss, and over 6% suffer from balance disorders. The cells responsible for sound and motion detections are the mechanosensory hair cells residing in the inner ear. As the degeneration of the hair cells is irreversible in mammals, there is currently no approved medications for sensory recovery. In recent years, derivation methods have been developed to generate inner ear cells from non-otic cells in vitro via stepwise morphogen treatment or forced activation of hair cell transcription factors (TFs). Despite providing unprecedented research opportunities, none of these current in vitro derivation approaches are suitable for high-throughput therapeutic discoveries due to limitations such as being difficult to scale up and relatively inefficient, or the lack of a spatially organized sensory epithelial structure. To overcome these limitations, this study aims to build a novel human inner ear organoid model by genetically converting aggregated human pluripotent stem cells (PSCs) into otic progenitor cells through CRISPR-based activation of otic progenitor TFs, followed by self-organized cellular maturation in 3D culture. A CRISPR screen will be performed to identify additional TFs that can enhance the lineage conversion efficiency. As only a single treatment step is required and the derivation protocol is otic lineage-focused, this new organoid model is expected to be more scalable and efficient than current stepwise morphogen treatment organoid models. Furthermore, as otic progenitors have been shown to be capable of autonomously generating properly organized sensory epithelium cell types in vitro, this novel organoid model is expected to harbor hair cells and supporting cells in a spatially organized manner, therefore better recapitulating the native sensory epithelium structures than the existing direct hair cell conversion models. Due to these advantages, this novel organoid model could potentially serve as a therapeutic discovery platform for screening compounds and testing gene therapy treatments for hearing loss and balance dysfunctions. In addition to establishing the new model, this study will investigate the underlying mechanism of otic differentiation by identifying the direct and indirect downstream genes of a subset of otic progenitor TFs. The identification of the transcription network that regulates the cellular identity transition from the otic progenitors towards mature sensory cell types will significantly advance our understanding of human inner ear development. Collectively, the proposed research will provide novel tools and insights in basic and translational inner ear research.