A CRISPR-Cas9-based high-throughput screen in human brain organoids of T-dark putative downstream effectors of SOX2 in neurodevelopmental disease

This project has been funded thanks to the Joint Call Fondazione Cariplo and Fondazione Telethon 2021

Mutation of the Sox2 gene causes defective development of multiple brain regions, leading to blindness, mental retardation, seizures. SOX2, a transcription factor, controls the activity of many genes, some of which are known to be involved, if mutated, in other neurodevelopmental defects. Understanding the molecular mechanisms of the brain defects is critical for attempts to develop novel therapies; it requires the knowledge of the roles of the genes controlled by SOX2. More broadly, identifying genes important for neural cells proliferation and for neuronal function in general would be important for understanding also other neurodevelopmental disorders, due to genes unrelated to SOX2, but involved in shared aspects of brain developmental programs. We identified over a thousand genes controlled by SOX2 by studies in neural cells cultured from neonatal mouse brain. Many human genes are homologous (roughly speaking, are the human version) to the mouse genes we identified. Many of them belong to the T-dark gene category, genes of which very little is known, which represents the focus of this Call. We chose 129 human T-dark genes, prioritizing those whose activity is more impaired in mouse neural cells in which the SOX2 gene has been eliminated. We assume that the reduced expression of these genes is likely to affect important neural cells functions. To identify those genes which have roles in neurodevelopment, we will use a screening procedure, that was recently developed to identify gene defects leading to microcephaly in humans, and that is based on growing in vitro human brain “organoids” derived from a previously established cell line. The screening is based on in vitro mutagenesis via the CRISPRCas9 recent methodology, and identifies the mutated genes by genomic sequencing. This technique previously identified the mutation, in microcephaly, of a gene whose activity can be rescued towards normality by an available pharmacological agent (ref. 19 in our project).