PROJECT SUMMARY Autism spectrum disorder (ASD) is a heterogeneous neurodevelopmental disorder with a complex genetic architecture. The development of effective therapeutics and diagnostic tools for ASD has been hindered by our incomplete understanding of underlying genetic variation. De novo variants (DNVs), estimated to contribute to 30-40% of cases, have been primarily studied in protein-coding regions of the genome. Hundreds of thousands of non-coding variants have been identified but deciphering their functional contribution to ASD etiology remains challenging. Cis-regulatory elements such as promoters and enhancers represent one avenue to assay the potential impact of non-coding DNVs, but their regulatory activity is dependent on cellular contexts such as cell type and activation state. The two cell types primarily involved in ASD biology are excitatory (glutamatergic) and inhibitory (GABAergic) neurons, both of which can be generated in vitro from human pluripotent stem cells and depolarized to model the transcriptomic and epigenetic changes caused by neuronal activation. Our lab annotated the enhancers present in both cell types at baseline and activated states and found 2495 enhancers containing non-coding ASD DNVs, including several hundred that are cell-type specific or activity-dependent. Using a massively parallel reporter assay (MPRA), this proposal will determine whether non-coding DNVs found in individuals with autism alter cis-regulatory activity in glutamatergic or GABAergic human neurons in either baseline or activated states. Further, gene-enhancer mapping has revealed that a subset of DNV-containing enhancers is predicted to regulate genes previously implicated in ASD. To validate cis-regulatory activity and to compare trans-effects on downstream gene networks, a CRISPR inhibition screen will be performed in both cell types and activation states, targeting 25 ASD genes and their DNV-containing enhancers. If successful, this work will demonstrate the potential functional contribution of non-coding de novo variants to ASD biology, which has thus far remained an outstanding question in the field. Moreover, this will generate transcriptomic datasets for top ASD risk genes in two clinically relevant cell types at both resting and active states to expand upon the growing number of functional genomics ASD studies, emphasizing convergent regulatory gene networks. This research will take place at the Icahn School of Medicine at Mount Sinai, containing the 2nd best NIH-funded neuroscience department and home to the Seaver Autism Center, renowned for bridging basic science and clinical trials for more effective ASD care. The scientific rigor, innovative techniques, sophisticated analyses, multi-disciplinary collaborations, and ample mentorship opportunities outlined here would propel me towards a successful career as an independent research professor studying the molecular mechanisms underlying psychiatric disorders while mentoring futur...