Arrayed single-gene perturbations identify drivers of human anterior neural tube closure

Abstract Genetic studies of human embryonic morphogenesis are constrained by ethical and practical challenges, restricting insights into developmental mechanisms and disorders. Human pluripotent stem cell (hPSC)–derived organoids provide a powerful alternative for the study of embryonic morphogenesis. However, screening for genetic drivers of morphogenesis in vitro has been infeasible due to organoid variability and the high costs of performing scaled tissue-wide single-gene perturbations. By overcoming both these limitations, we developed a platform that integrates reproducible organoid morphogenesis with uniform single-gene perturbations, enabling high-throughput arrayed CRISPR interference (CRISPRi) screening in hPSC-derived organoids. To demonstrate the power of this platform, we screened 77 transcription factors in an organoid model of anterior neurulation to identify ZIC2, SOX11, and ZNF521 as essential regulators of neural tube closure. We discovered that ZIC2 and SOX11 are required for closure, while ZNF521 prevents ectopic closure points. Single-cell transcriptomic analysis of perturbed organoids revealed co-regulated gene targets of ZIC2 and SOX11 and an opposing role for ZNF521, suggesting that these transcription factors jointly govern a gene regulatory program driving neural tube closure in the anterior forebrain region. Our single-gene perturbation platform enables high-throughput genetic screening of in vitro models of human embryonic morphogenesis. Competing Interest Statement S.R., G.A., and R.H. are authors on the following patent application, which contains aspects of this work: Bioengineering and machine learning framework for complex tissue development. Application serial number PCT/US24/28838. Footnotes This manuscript has been updated to address reviewer comments. Figures 1 and S1 have been updated to include more thorough analyses of timepoints from in vivo human neural data. The Figure 1 caption has been updated to specify geometry of organoids and our interpretation of lumen stain data. Two supplementary videos have been added showing shedding of dead cells into the lumen. The Methods section has been updated to include details of mediolateral axis analysis from scRNA-seq data. https://figshare.com/articles/dataset/2026_HuangAnand/31151509