Human brain development involves intricate processes, such as precise gene regulation and the formation of complex cytoarchitectures, which are challenging to study due to their inherent heterogeneity. To address this, we integrated CRISPR-based screening with stem cell-derived cerebral organoids to develop a new platform that can test gene function in this complex environment. We first analysed data from neuro-specific whole-genome knockout screens in Drosophila, and identified 129 human genes of undefined function. To screen these genes, we developed ORIGUMI (ORganoid Interrogation by Guides and Unique Molecular Identifiers), a CRISPR knockout system that employs genetic barcodes to enable high-throughput screening in heterogeneous tissues like brain organoids. ORIGUMI successfully identified controls predicted for depletion (e.g., SOX2) or enrichment (e.g., SUFU), while providing novel targets currently under study. We were able to validate a majority of these hits using two independent CRISPR-based approaches: (1) by using individual CRISPR-KOs and quantifying gross organoid size, and (2) by tracking fluorescently labelled CRISPR-knockout cells throughout development. This work demonstrates that ORIGUMI is a robust tool for gene discovery in three-dimensional brain tissues and offers new avenues for studying neurological disorders, including autism and schizophrenia. Our findings lay the groundwork for future collaborations at the intersection of genomics, stem cell biology, and brain research.