A CRISPR loss-of-function Hepatic progenitor cells expansion screen in liver organoids

Biliary epithelial cells (BECs) form bile ducts in the liver, and are facultative liver stem cells that establish a ductular reaction (DR) to support liver regeneration following injury. Liver damage induces periportal LGR5+ putative liver stem cells which can form BEC-like organoids, suggesting RSPO-LGR4/5-mediated WNT/β-catenin activity is important for a DR. We addressed the roles for this and other signaling pathways in a DR by performing a focused CRISPR-based loss-of-function screen in BEC-like organoids, followed by in vivo validation and single-cell RNA sequencing. We found BECs lack and do not require LGR4/5-mediated WNT/β-Catenin signaling during DR, while YAP and mTORC1 signaling are required for this process. Upregulation of LGR5/AXIN2 is required in hepatocytes to enable their regenerative capacity in response to injury. Together, these data highlight heterogeneity within the BEC pool and delineate signaling pathways involved. The 1000 element library is a pooled CRISPR library with 1000 unique CRISPR sgRNA. The take rate library is a DNA barcode library (https://www.cellecta.com/content/uploads/2014/10/Cellecta-PAC-Barcode-Library-13Kx13K-Plasmid-12082901.pdf). The manuscript presents the finding of a pooled CRISPR screen on liver organoid culture system using a 1000 element CRISPR library. To develop the screening assay, we evaluated the clonal growth kinetics of the liver organoids by infecting the organoids with the take rate library which is composed of DNA barcodes that do not perform genetic perturbation. The take rate library result confirms that the liver organoids do not display strong clonal growth and thus are suitable for screening. We use cell survival and proliferation as the measure for identifying signalling pathways regulating HPC expansion in vitro.