Lentiviral SgRNA Delivery for CRISPR/Cas9 Editing in Cre-Dependent Cas9 Knock-in Primary Mouse Hepatocytes

Over the past few decades, CRISPR/Cas9-mediated genome editing has transformed functional studies in cell line models by making genetic manipulation highly efficient and feasible. However, extending this technology to primary hepatocytes remains a major challenge. While cultured primary hepatocytes are indispensable for disease modeling and drug development as they retain key metabolic functions absent in cell lines, their limited in vitro lifespan and negligible proliferative capacity pose fundamental barriers to efficient genome editing. Here, we describe an effective two-step perfusion protocol that enables the isolation of primary hepatocytes with high cell viability (>90%) and high yield (approximately 1 × 10⁷ hepatocytes per adult mouse). Following isolation, lentiviral sgRNA transduction is typically performed within 3-4 h, and genome editing outcomes are assessed 5-7 days post-infection. Using hepatocytes from transgenic LSL-Cas9-EGFP mice, in which the Cas9 cassette was activated by a lentiviral vector co-expressing Cre recombinase and sgRNA, achieving up to 80% allele-level gene knockout efficiency in monolayer cultures. In addition, we achieved approximately 12% gene KO efficiency in three-dimensional hepatocyte organoids (HEOs), which more closely recapitulate the architecture and functional characteristics of native liver tissue. In this protocol, a successful experiment is defined by three criteria: sufficient hepatocyte yield (>5 × 10⁶ cells per mouse) with high viability (>85%), efficient single-guide RNA (sgRNA) delivery into hepatocytes, and validated target gene disruption at the genomic level. This protocol demonstrates the feasibility of efficient in vitro genome manipulation in both monolayer-cultured hepatocytes and HEOs, providing a robust platform for genetic modeling and functional studies of liver diseases.