利用肝细胞进行基础研究和再生医学需要完全了解肝细胞分化和成熟的遗传决定因素。类器官中的单细胞 CRISPR 筛选可以将遗传扰动与单细胞中的平行转录组读数联系起来，为描述细胞命运调节剂的作用提供了一种强大的方法。然而，在数据分析过程中识别关键调控因子的一大挑战是转录因子（TF）的低表达水平，由于单细胞测序中的噪音和丢失，很难准确估计。此外，与细胞命运转变相关的往往是转录级联中 TF 活性的变化，而不是 TF 的表达水平。在这里，我们开发了基于类器官的单细胞 CRISPR 筛选分析调节子 (OSCAR)，一种使用调节子活动作为读数来剖析类器官中基因敲除效应的框架。在成体干细胞衍生的肝脏类器官中，我们在与肝细胞形成转录调控相关的 246 个扰动下绘制了 80,576 个细胞的转录组图谱。使用 OSCAR，我们确定了已知的和新颖的正负调节因子，其中 Fos 和 Ubr5 是排名最高的调节因子。进一步的单基因功能丧失测定表明，小鼠和人类肝脏类器官中的 Fos 耗竭通过肝脏代谢基因和途径的特异性上调促进肝细胞分化，并且小鼠肝脏中 Ubr5 的条件性敲除延迟了肝细胞成熟。总之，我们提供了框架以快速、系统的方式探索谱系说明符，并识别具有潜在临床应用的肝细胞决定因子。© 2023。作者。

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.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.Altogether, we provide a framework to explore lineage specifiers in a rapid and systematic manner, and identify hepatocyte determinators with potential clinical applications.© 2023. The Author(s).