Loss-of-function (LOF) screens provide a powerful approach to identify regulators in biological processes. Pioneered in laboratory animals, LOF screens of human genes are currently restricted to two-dimensional (2D) cell culture hindering testing of gene functions requiring tissue context. Here we present CRISPR-LIneage tracing at Cellular resolution in Heterogenous Tissue (CRISPR-LICHT), enabling parallel LOF studies in human cerebral organoid tissue. We used CRISPR-LICHT to test 173 microcephaly candidate genes revealing 25 to be involved in known and uncharacterized microcephaly-associated pathways. We characterized Immediate Early Response 3 Interacting Protein 1 (IER3IP1) regulating the unfolded protein response (UPR) and extracellular matrix (ECM) protein secretion crucial for tissue integrity, with dysregulation resulting in microcephaly. Our human tissue screening technology identifies microcephaly genes and mechanisms involved in brain size control.

Pluripotency can be captured in vitro in the form of Embryonic Stem Cells (ESCs). These ESCs can be either maintained in the unrestricted "naïve" state of pluripotency, adapted to developmentally more constrained "primed" pluripotency or differentiated towards each of the three germ layers. Epigenetic protein complexes and transcription factors have been shown to specify and instruct transitions from ESCs to distinct cell states. In this study, proteomic profiling of the chromatin landscape by chromatin enrichment for proteomics (ChEP) is used in mouse naive pluripotent ESCs, primed pluripotent Epiblast stem cells (EpiSCs), and cells in early stages of differentiation. A comprehensive overview of epigenetic protein complexes associated with the chromatin is provided and proteins associated with themaintenance and loss of pluripotency are identified. The data reveal major compositional alterations of epigenetic complexes during priming and differentiation of naïve pluripotent ESCs. These results contribute to the understanding of ESC differentiation and provide a framework for future studies of lineage commitment of ESCs.

Summary Retinoic acid (RA) signaling is an important and conserved pathway that regulates cellular proliferation and differentiation. Furthermore, perturbed RA signaling is implicated in cancer initiation and progression. However, the mechanisms by which RA signaling contributes to homeostasis, malignant transformation, and disease progression in the intestine remain incompletely understood. Here, we report, in agreement with previous findings, that activation of the Retinoic Acid Receptor and the Retinoid X Receptor results in enhanced transcription of enterocyte-specific genes in mouse small intestinal organoids. Conversely, inhibition of this pathway results in reduced expression of genes associated with the absorptive lineage. Strikingly, this latter effect is conserved in a human organoid model for colorectal cancer (CRC) progression. We further show that RXR motif accessibility depends on progression state of CRC organoids. Finally, we show that reduced RXR target gene expression correlates with worse CRC prognosis, implying RA signaling as a putative therapeutic target in CRC.