Genome-wide CRISPRi Screen in Human iNeurons to Identify Novel Focal Cortical Dysplasia Genes

Abstract

AbstractFocal cortical dysplasia (FCD) is a common cause of focal epilepsy that typically results from brain mosaic mutations in the mTOR cell signaling pathway. To identify new FCD genes, we developed anin vitroCRISPRi screen in human neurons and used FACS enrichment based on the FCD biomarker, phosphorylated S6 ribosomal protein (pS6). Using whole-genome (110,000 gRNAs) and candidate (129 gRNAs) libraries, we discovered 12 new genes that significantly increase pS6 levels. Interestingly, positive hits were enriched for brain-specific genes, highlighting the effectiveness of using human iPSC-derived induced neurons (iNeurons) in our screen. We investigated the signaling pathways of six candidate genes:LRRC4,EIF3A, TSN, HIP1, PIK3R3,andURI1. All six genes increased phosphorylation of S6. However, only two genes,PIK3R3andHIP1,caused hyperphosphorylation more proximally in the AKT/mTOR/S6 signaling pathway. Importantly, these two genes have recently been found independently to be mutated in resected brain tissue from FCD patients, supporting the predictive validity of our screen. Knocking down each of the other four genes (LRRC4, EIF3A, TSN, and URI1) in iNeurons caused them to become resistant to the loss of growth factor signaling; without growth factor stimulation, pS6 levels were comparable to growth factor stimulated controls. Our data markedly expand the set of genes that are likely to regulate mTOR pathway signaling in neurons and provide additional targets for identifying somatic gene variants that cause FCD.Significance StatementFocal cortical dysplasia (FCD) is a common cause of focal epilepsy due to somatic variants in mTOR pathway genes in FCD brain tissue. Unbiased sequencing to identify novel FCD genes is challenging since these variants are often in a small subset of neurons. To overcome this challenge, we used anin vitrogenetic screen in human neurons using an FCD biomarker, uncovering genes that increase neuronal mTOR signaling when their expression is lost. Two candidate genes were mutated in patient FCD brain tissues in a recent study, supporting a causative role for these genes in FCD. Our work suggests gene candidates for somatic variant analysis in FCD tissue and indicates added value for genetic screening in neurons over cell lines.