Project Abstract Autism Spectrum Disorder (ASD) is the most common neurodevelopmental disorder, yet the neurobiological mechanisms underlying ASD pathogenesis remain largely unknown. Large-scale exome sequencing studies of individuals with ASD have identified over 100 genes significantly associated with ASD risk. Functional characterization of ASD risk genes can provide insight to ASD pathogenesis. We and others have recently identified de novo loss-of-function variants in the transcription factor RFX3 as a relatively common monogenic cause of ASD, implying an important role for RFX3 in human neurodevelopment. We have found evidence that RFX3 may be a critical transcriptional regulator of the development and function of layer II/III neurons: its expression is significantly enriched in cortical layer II/III excitatory neurons, and the RFX3 binding motif is specifically enriched in accessible chromatin regions of the human fetal germinal zone and layer II/III excitatory neurons. In this proposed research, I will address the hypothesis that RFX3 regulates key neurodevelopmental processes in layer II/III excitatory neurons and the expression of other ASD risk genes that affect neuronal formation and function. In Aim 1, I will identify the genes and pathways regulated by RFX3 in human cortical neurons by profiling the genome-wide binding sites of RFX3 and the transcriptional changes induced by loss of RFX3 occupancy in RFX3 haploinsufficient human iPSC-derived neurons. In Aim 2, I will evaluate the effect of RFX3 haploinsufficiency on cortical neuron formation and synaptic function in human iPSC-derived forebrain organoids. I will use single-cell RNA-sequencing to identify changes in cell type composition and infer alterations in developmental trajectories in RFX3 deficient organoids, and multielectrode array to assess synaptic plasticity balance in RFX3 deficient organoids compared to isogenic controls. Taken together, this proposal will yield insight on the transcriptional programs regulated by RFX3 in human neurons, and how RFX3 haploinsufficiency disrupts neuronal development and function. This will allow for improved understanding of ASD neurobiology, and the development of novel targeted therapies for ASD.