Fig. 1

Establishment of an organoid-based single-cell CRISPR screen for hepatocyte lineage regulators. a Schematic design of the organoid-based CROP-seq screen. b Mouse ICOs isolated from Cas9-EGFP knockin mice were transduced with the pilot CROP-seq screen lentiviral library carrying a mCherry reporter (pre-FACS; top panel). At day 3 post-transduction, mICOs were enriched with FACS for genome-edited cells (GFP⁺/mCherry.⁺) and expanded (post-FACS; bottom panel). Scale bars, 500 μm. c The number and corresponding ratio of cells expressing a unique sgRNA (red), more than two sgRNAs (green) or none (blue) in the EM and DM groups. d UMAP visualization of cells with unique sgRNA assignments in the EM and DM groups. e UMAP visualization of the expression of known hepatic markers, including Alb, Ttr, Mup20, Apoa1, Sult1a1, and Aldh1a1. Expression is color-scaled in log2 for each cell. f Heatmap graph displaying the perturbation effect of each sgRNA on the top 20 variable genes. g Violin plots showing the relative expression of known hepatocyte and cholangiocyte markers in Hnf4α-KO single cells in the pilot screen. Color filling indicates a P-value less than 0.05 (Wilcoxon rank sum test)