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

Focal 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 an CRISPRi screen in human neurons and used FACS enrichment based on the FCD biomarker, phosphorylated S6...

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Bibliographic Details
Published inbioRxiv : the preprint server for biology
Main Authors Tidball, Andrew M, Luo, Jinghui, Walker, J Clayton, Takla, Taylor N, Carvill, Gemma L, Parent, Jack M
Format Journal Article
LanguageEnglish
Published United States 13.12.2023
Subjects
epilepsy
focal cortical dysplasia
iNeuron
CRISPRi screen
mTOR
Biological sciences
Neuroscience/Genetics
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Summary:Focal 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 an CRISPRi 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: , and . All six genes increased phosphorylation of S6. However, only two genes, and 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 ( ) 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.