PROJECT SUMMARY A complex interplay of genetic variation underlies predisposition for autism spectrum disorder (ASD). There is now strong evidence from large consortia studies that mutations in genes involved in chromatin modification, transcriptional regulation, and synaptic proteins confer substantial risk for ASD; however, the extent to which these genes are interconnected and ultimately converge on a small number of functional deficits is largely unknown. A critical need therefore exists to model new gene discoveries, to directly evaluate their functional impact, and to determine their points of convergence. Innovations from our team and others in high-throughput CRISPR-engineering have now made parallelized mechanistic studies tractable, and human induced pluripotent stem cell (hiPSCs) derived neurons are well-suited to test the impact of ASD risk variants predicted to exert their influence during fetal cortical development. Here, our multi-PI proposal will undertake an ambitious, systematic isogenic loss-of-function (LoF) mechanistic screen in a compendium of 48 of the most robust ASD risk genes discovered from the largest genetic studies to date. Moreover, our exciting preliminary results suggest that transcriptional signatures shared across neuronal models of ASD genes converge on critical regulatory nodes that result in synaptic deficits. Aim 1 will characterize isogenic glutamatergic and GABAergic neurons with highly penetrant LoF mutations in 48 genes associated with ASD risk at genome-wide significant thresholds and that are expressed in neurons. These analyses will identify transcriptional and functional signatures of individual ASD genes through RNAseq and a series of high-throughput phenotyping assays in both neuronal sub-types. Aim 2 will expand our Preliminary Results to discover convergent genes downstream of ASD risk loci, characterize the synaptic consequences of the ten most compelling discoveries from individual genes and/or convergent signatures, and integrate these data to explore the druggability of the convergent networks. Our overarching goal is to define any commonalities among diverse genes, pathways and networks that underlie ASD etiology, and to dramatically expand the list of possible therapeutic targets for ASD. These studies will generate an unprecedented isogenic resource of CRISPR-edited ASD genes, and matched RNAseq and cellular phenotyping in glutamatergic and GABAergic neurons, that will be provided for open distribution to the broader community through the NIMH RUDCR resource to yield new insights into neuropsychiatric disorders.