High-throughput single-cell CRISPRi screens stratify neurodevelopmental functions of schizophrenia-associated genes

Abstract Schizophrenia is a complex neuropsychiatric disorder with strong genetic underpinnings, yet the molecular mechanisms linking genetic risk to disrupted brain development remain poorly understood. Transcription factors (TFs) and chromatin regulators (CRs) are increasingly implicated in neuropsychiatric disorders, where their dysregulation may disrupt neurodevelopmental programs. Despite this, systematic functional interrogation in human models has been limited. Here, we combine pooled CRISPR interference (CRISPRi) screens with high- throughput single-cell multiomic profiling in hiPSC-derived neural progenitors and neurons to functionally assess 65 schizophrenia-associated genes. Based on public datasets and literature review, we selected 55 TFs and CRs, along with ten additional risk genes whose loss-of-function has been linked to schizophrenia. Our single-cell CRISPRi readouts revealed that perturbations in TFs and CRs converge on disrupting neurodevelopmental timing. CRISPRi of several factors delayed neural differentiation, whereas others, such as the knockdown of MCRS1, drove precocious neural commitment. Validation screens combined with cell cycle and metabolic indicators confirmed the differentiation-restricting or -promoting roles of these TFs and CRs. Multimodal trajectory analysis uncovered discrete transcriptional and epigenomic states representing delayed and accelerated neurodevelopment, enriched for schizophrenia GWAS loci and disease-relevant pathways. Gene regulatory network (GRN) inference identified TCF4 and ZEB1 as critical mediators opposing the neural differentiation trajectory. Functional overexpression of these TFs followed by chromatin profiling demonstrated that TCF4 restrains, while ZEB1 promotes, neural differentiation in a stage-specific and competitive manner. Furthermore, we show that MCRS1 represses ZEB1 expression, positioning MCRS1 as a key brake on premature neurodevelopment. Together, our study establishes a scalable framework that integrates genetic perturbation, single- cell multiomics, and GRN modeling to functionally annotate disease-linked genes. We reveal convergent regulatory axes that underlie altered neurodevelopmental timing in schizophrenia, offering mechanistic insights into how chromatin misregulation contributes to disease pathogenesis. Competing Interest Statement The authors have declared no competing interest.