In-organoid single-cell CRISPR screening reveals determinants of hepatocyte differentiation and maturation

BACKGROUND: Harnessing hepatocytes for basic research and regenerative medicine demands a complete understanding of the genetic determinants underlying hepatocyte differentiation and maturation. Single-cell CRISPR screens in organoids could link genetic perturbations with parallel transcriptomic readout in single cells, providing a powerful method to delineate roles of cell fate regulators. However, a big challenge for identifying key regulators during data analysis is the low expression levels of transcription factors (TFs), which are difficult to accurately estimate due to noise and dropouts in single-cell sequencing. Also, it is often the changes in TF activities in the transcriptional cascade rather than the expression levels of TFs that are relevant to the cell fate transition. RESULTS: Here, we develop Organoid-based Single-cell CRISPR screening Analyzed with Regulons (OSCAR), a framework using regulon activities as readouts to dissect gene knockout effects in organoids. In adult-stem-cell-derived liver organoids, we map transcriptomes in 80,576 cells upon 246 perturbations associated with transcriptional regulation of hepatocyte formation. Using OSCAR, we identify known and novel positive and negative regulators, among which Fos and Ubr5 are the top-ranked ones. Further single-gene loss-of-function assays demonstrate that Fos depletion in mouse and human liver organoids promote hepatocyte differentiation by specific upregulation of liver metabolic genes and pathways, and conditional knockout of Ubr5 in mouse liver delays hepatocyte maturation. CONCLUSIONS: Altogether, we provide a framework to explore lineage specifiers in a rapid and systematic manner, and identify hepatocyte determinators with potential clinical applications. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13059-023-03084-8.