Scaled and efficient derivation of loss-of-function alleles in risk genes for neurodevelopmental and psychiatric disorders in human iPSCs

Translating genetic findings for neurodevelopmental and psychiatric disorders (NPDs) into actionable disease biology would benefit from large-scale and unbiased functional studies of NPD genes. Leveraging the cytosine base editing (CBE) system, we developed a pipeline for clonal loss-of-function (LoF) allele mutagenesis in human induced pluripotent stem cells (hiPSCs) by introducing premature stop codons (iSTOP) that lead to mRNA nonsense-mediated decay (NMD) or protein truncation. We tested the pipeline for 23 NPD genes on 3 hiPSC lines and achieved highly reproducible, efficient iSTOP editing in 22 genes. Using RNA sequencing (RNA-seq), we confirmed their pluripotency, absence of chromosomal abnormalities, and NMD. Despite high editing efficiency, three schizophrenia risk genes ( SETD1A, TRIO, and CUL1) ) only had heterozygous LoF alleles, suggesting their essential roles for cell growth. We found that CUL1-LoF LoF reduced neurite branches and synaptic puncta density. This iSTOP pipeline enables a scaled and efficient LoF mutagenesis of NPD genes, yielding an invaluable shareable resource.