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

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 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 , , and as essential regulators of neural tube closure. We discovered that and are required for closure, while prevents ectopic closure points. Single-cell transcriptomic analysis of perturbed organoids revealed co-regulated gene targets of and and an opposing role for , 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 models of human embryonic morphogenesis.