Enhancer-targeted correction of haploinsufficient autism risk genes

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Project Summary/Abstract Autism Spectrum Disorder (ASD) is a highly heterogeneous and highly heritable disease with complex genetic contributions, and yet about 20% of genetic risk is imparted by de novo, or newly arising, mutations of major effect. The majority of mutations in these genes are either known to or predicted to lead to truncated mRNA and protein products, strongly indicating likely haploinsufficiency, in which a single copy of a functional gene is not sufficient for normal brain development or function. I propose a carefully staged approach to correct the effects of these mutations by activating expression from the non-mutant copy to restore gene expression to normal levels. I will leverage advances in gene editing technology - using CRISPR-A to target enhancer regions of high confidence ASD genes in stem cell-based 3D cortical spheroids (hCS) in vitro, which have been shown to recapitulate many features of in vivo cortex development. CRISPR-A has the significant advantages of leveraging endogenous gene expression regulatory elements without editing the genome or relying on artificial gene constructs for overexpression. I also leverage previous work where we have created high-confidence genome-wide enhancer maps that connect these transcriptional activating regions with their cognate genes. I propose to: i) functionally validate putative enhancer sequences for 12 high confidence ASD genes, ii) test selected enhancers in hCS to characterize their effect on neuronal differentiation, and iii) characterize changes in developmental and morphological phenotypes in hCS carrying ASD risk mutations compared to wildtype hCS, and determine the ability of CRISPR-A to rescue gene expression and restore wildtype development in mutant hCS. I will utilize diverse technologies, including single cell sequencing and CLARITY imaging, to comprehensively visualize the development of neuronal subtypes in cortical spheroids. Results from this study may not only provide a proof of principle for gene activation as a therapeutic intervention but will substantially enhance our understanding of the effects of haploinsufficiency in ASD genes and the neurobiological mechanisms whereby they impact neuronal development.