High throughput genomic edition to investigate neurodevelopmental disorders using isogenic cellular models

Abstract

The genetic architecture of neurodevelopmental disorders (NDD) includes rare, highly penetrant mutations and common variants of smaller effect. De novo, rare loss-of-function (LoF) and missense mutations in evolutionarily constrained genes are the strongest known risk factors for NDD, and these genes are particularly enriched in two main pathways: (1) neuronal communication and (2) regulation of gene expression, i.e., chromatin regulators and transcription factors. Functional analyses suggest that these two pathways are interconnected during neurodevelopment; however, their mechanisms of convergence are unknown. Moreover, the relative impact of LoF variants compared to missense mutations is poorly understood. In this proposal, we will leverage a CRISPR-based high throughput genomic engineering platform for parallelized genome edition in human induced pluripotent stem cell (hiPSC) lines to introduce LoF and missense perturbations on NDD-associated genes related to transcription regulation. hiPSC isogenic cellular models will be generated and differentiated in neural stem cells and glutamatergic cortical neurons to interrogate the molecular consequences the genomic editions created. We will explore direct, or proximal, dysregulation of critical neuronal genes following the LoF of these transcription regulators (Aim 1), and then compare the relative impact of patient-specific missense variants vs. gene deletions (Aim 2). These hiPSC-derived neuronal lineages will have their transcriptome and chromatin accessibility profile defined by RNA-seq and Assay for Transposase-Accessible Chromatin (ATAC)-seq to infer the molecular consequences followed by the introduction of these genomic variants. This study will thus leverage new technologies, neuronal models, and complementary datasets to identify the nodes of coalescence in molecular signatures of NDD, which may correspond to druggable targets in future studies. (AU)