Genetic editing in induced pluripotent stem cells for the production of isogenic neural models with variants in POGZ for the study of neurodevelopmental disorders

Abstract

CRISPR/Cas9-based genomic engineering allows for simultaneous genome edits to introduce different pathogenic genetic variants and decipher the connection points between their functional consequences. The application of this genomic editing platform in human induced pluripotent stem cell (hiPSC) lines can lead to the investigation of genes and cell types associated with neurodevelopmental disorders (NDD). Several chromatin regulatory genes have been implicated in autism spectrum disorder (ASD). Among them, POGZ has particularly strong statistical evidence for association with NDD and ASD, driven mainly by rare loss-of-function variants. Despite its clinical relevance, little is known about the regulatory mechanisms of POGZ during neurodevelopment. Detecting the functional impacts of the nonsense variant in POGZ observed in patients with NDD/ASD involves evaluating the molecular signatures in neuronal models derived from hiPSC. We will use applications that encompass three methodologies: (1) hiPSC culture, (2) hiPSC genetic editing via CRISPR/Cas9, and (3) neuronal differentiation from hiPSC carrying the genetic editing. With CRISPR/Cas9, we will introduce a nonsense variant in the POGZ gene. Isogenic hiPSC models with this POGZ variant and control lines will be differentiated into neural stem cells (NSC). The neuronal lines derived from hiPSCs carrying genomic edits will have their transcriptome delineated via RNA-seq, aiming to define the molecular consequences derived from the introduction of this genetic variant. The molecular profile of the obtained neuronal models will be contrasted with RNA-seq databases available in the literature generated from animal models with mutations in Pogz. The convergent molecular nodes observed will be interpreted against the neurological alterations observed in patients with variants in this gene, providing future perspectives for treatments with the application of precision medicine, offering efficient drug options.