A CRISPR screen in human iPSC-derived neurons reveals convergent transcriptomic effects of neurodevelopmental disorder genes

Diverse risk genes have been identified for neurodevelopmental disorders (NDDs), but how these genes converge on similar biological pathways in neurons, and thus give rise to similar phenotypes, is unclear. Here, we apply a pooled CRISPR approach to target 23 (out of 29) NDD loss-of-function genes and examine convergent effects on gene expression across human induced pluripotent stem cell (hiPSC)-derived neural progenitor cells, glutamatergic neurons, and GABAergic neurons. Points of convergence vary between these cell-types and are greatest in mature glutamatergic neurons, where they broadly regulate synaptic, epigenetic, and, unexpectedly, mitochondrial pathways. The strongest convergent networks were observed between NDD genes with shared biological annotations, clinical associations, and co-expression patterns in human post-mortem brain. Drugs that were predicted to reverse convergent transcriptomic signatures and/or arousal and sensory processing behaviors ameliorated behavioral phenotypes in NDD model zebrafish. These results suggest that convergent effects of NDD risk genes could provide clinically useful insights. Code and secondary analysis data can be found on Synapse ( ID=syn72039767, link=https://www.synapse.org/Synapse:syn72039767/wiki/636167) Overall design: Transient transcription factor overexpression from stable clonal hiPSCs was used to induce control hiPSCs to iNPCs (here SNaPs), Lentiviral Cas9v2-HygroR and pooled LentiArray-gRNA-PuroR CRISPR-KO library including 29 genes (ARID1B, ASH1L, CHD2, MED13L, NRXN1, PHF21A, SETD5, SIN3A, SMARCC2, BCL11A, CHD8, KDM5B, KDM6B, KMT2C, MBD5, PHF12, SKI, WAC, CREBBP, ASXL3, ANK3, POGZ, SCN2A, SHANK3, SLC6A1, PPP2R5D, FOXP2, DPYSL2) as well as scrambled gRNA were packaged as high-titer lentiviruses were transduced via spinfection three days prior to harvest (e.g., d4 for D7 iGLUTs, d18 for D21 iGLUTs, d33 for d36 iGABA) (MOI 0.3-0.5). The entire cell were then submitted for single cell RNAseq (10X single cell 5' v2 HT with CRISPR barcode kit) in four lanes per cell type, targeting 20,000 cells per lane for a total of ~80,000 targeted cells per cell type, at Yale Genomics Core.