Modified Project Summary/Abstract Section Rare loss-of-function (LoF) mutations in SETD1A are strongly associated with schizophrenia (SZ), a debilitating mental disorder affecting 1% of the population, and other severe neurodevelopmental disorders. SETD1A encodes a component of the histone methyltransferase complex producing mono-, di, and trimethylated histone H3 at Lysine 4 (H3K4). H3K4 trimethylation (H3K4me3) and H3K4me1 are epigenomic marks of active gene transcriptional promoters and enhancers, respectively. Interestingly, histone methylation has also been suggested as one of the most enriched gene pathways in common variant-based genome-wide associations studies (GWAS) of major psychiatric disorders. Furthermore, a recent mouse model with heterozygous knockout of SETD1A exhibited working memory deficits and showed transcriptional changes that overlap with those implicated in neurodevelopmental disorders, however, seemingly independent from a H3K4me3 mechanism. Therefore, it remains largely unclear whether and how SETD1A causes SZ-relevant molecular and cellular changes in a human brain. Our central hypothesis is that human induced pluripotent stem cell (hiPSC)-derived neuronal cells and cortical organoids recapitulate key SZ-relevant epigenetic, molecular and cellular properties of SETD1A LoF in the human brain. Using CRISPR/Cas9 gene editing, we have generated isogenic hiPSC lines carrying heterozygous LoF mutations (in exon 4 and exon 16, on different genetic backgrounds) of SETD1A. Preliminary results showed that mutant lines were defective in cortical organoid development with premature neuronal differentiation at early developmental stages. Furthermore, morphological, electrophysiological and transcriptomic analyses of hiPSC neurons carrying SETD1A LoF mutation showed defective synaptic neurotransmission. Interestingly, genes showing differential expression in both 3D cortical organoids and 2D cultures from mutant lines are enriched for common GWAS risk variants of SZ and other neuropsychiatric disorders/traits, suggesting possible convergent pathways shared by SETD1A LoF and common GWAS risk variants of major psychiatric disorders. Leveraging our respective expertise in hiPSC models and neurogenesis, synaptic physiology and functional genomics within our team, we propose to characterize the molecular and cellular mechanisms underlying the deficits associated with SZ-associated LoF mutations in SETD1A in human neural systems. We will identify the cell-type-specific and developmental stage-specific cellular and molecular phenotypes associated with SETD1A LoF in cortical organoids, and then investigate the synaptic phenotype(s) of SETD1A LoF mutations in human neurons and associated transcriptome changes. The proposed study will enable us to perform a well-controlled assessment of the impact of SETD1A LoF mutations on the molecular and cellular mechanisms underlying deficits in early neurodevelopment and synaptic properties.