PROJECT SUMMARY Pathogenic variants in CHD2 are associated with intellectual and developmental disabilities (IDDs), including autism spectrum disorder, developmental delay, and early onset epilepsy. Chromodomain helicase DNA binding protein 2 (CHD2) is a chromatin remodeling protein that alters chromatin structure, leading to changes in gene expression of downstream targets. Little is known about how variants in CHD2 lead to IDDs. The mouse model of Chd2 deficiency does not recapitulate the seizure phenotype seen in human patients with CHD2 variants, indicating that there may be species-specific functions of CHD2 in the brain. I hypothesize that CHD2 will be important for regulating genes involved in the development of primate-specific brain structures, such as the outer subventricular zone. To study the mechanism of CHD2 in a human neuronal system, we propose the development of a 3D cerebral organoid (CO) model of CHD2 deficiency. Using CRISPR/Cas9, we have genetically engineered an induced pluripotent stem cell (iPSC) line carrying an endogenous form of CHD2 with Flag, HA, and green fluorescent protein (GFP) tags (WTCHD2-Flag-HA-GFP). In our first aim, we propose deletion of one copy of CHD2 using CRISPR/Cas9, resulting in iPSCs haploinsufficient for CHD2 (CHD2+/-Flag- HA-GFP). WTCHD2-Flag-HA-GFP iPSCs and CHD2+/-Flag-HA-GFP iPSCs will then be differentiated into forebrain specific COs. I will determine neurodevelopmental defects upon CHD2 disruption, such as differences in neuronal differentiation, proliferation, and formation of distinct cerebral cortex layers, through a combination of immunohistochemistry and cell cycle analyses. In Aim 2 I will identify genes misregulated upon CHD2 disruption, by performing single-cell mRNA sequencing in WTCHD2-Flag-HA-GFP and CHD2+/-Flag-HA-GFP COs. In Aim 3, I will determine genome-wide CHD2 occupancy, by performing chromatin immunoprecipitation followed by next generation sequencing (ChIP-Seq) on cells dissociated from WTCHD2-Flag-HA-GFP COs. I will then correlate the results of CHD2 occupancy with the data on which genes are misregulated upon CHD2 disruption to identify a set of candidate genes that are regulated by CHD2 in human neuronal cells. Combined, these studies will lead to identification of CHD2-regulated genes in human neuronal cell types, revealing pathways in which CHD2 is involved and improving our understanding of the biological mechanisms behind CHD2- associated IDDs. In the future this model has the potential for drug development by attempting to rescue defects with small molecules.