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

Summary

This protocol describes the isolation and culture of primary hepatocytes from Loxp-Stop-Loxp (LSL)-Cas9-EGFP mice and lentiviral sgRNA delivery for CRISPR/Cas9-mediated gene knockout in monolayer cultures and hepatocyte organoids. The method enables high hepatocyte yield and viability and supports efficient gene-specific knockout validation in physiologically relevant primary hepatocyte models.

Abstract

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.

Introduction

Genome-editing technologies enable precise investigation of gene function in physiologically relevant systems. Therefore, applying CRISPR/Cas9 directly to primary cells is a critical strategy, as it enables genetic investigation within a native cellular environment^1,2. For instance, this approach has enabled the modeling of cystic fibrosis in primary intestinal stem cells³, the study of Duchenne muscular dystrophy in patient-derived myoblasts⁴, and the therapeutic engineering of primary T cells for cancer immunotherapy⁵.

Protocol

Ethics statement
All animal procedures were performed in accordance with institutional guidelines and were approved by the Institutional Animal Care and Use Committee (IACUC) of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences (Approval No. 2024-12-ZDX-010).

Figure 1 provides a schematic overview of the experimental workflow.

1. Construction of sgRNA plasmid

1. Order a pair of forward and reverse oligos for generating the sgRNA cloning fragment.
   Oligo_gSP_F: TATATATCTTGTG
   GAAAGGACGAAACACCGNNNNNNNNNNNN....

Results

Figure 1 presents a schematic overview of the experimental workflow for lentivirus-mediated CRISPR/Cas9 gene editing in primary hepatocytes cultured in monolayer and HEO formats.

To evaluate whether the HCM supports monolayer-cultured hepatocytes for gene-editing applications in vitro, we monitored cell morphology, the expression of the hepatic metabolic marker Cytochrome P450 3A4 (CYP3A4) and cell viability over time (Figure 2

Discussion

This protocol provides an efficient, cost-effective, and reproducible workflow for CRISPR/Cas9-mediated genome editing in both monolayer-cultured mouse hepatocytes and HEOs, achieving high genome-editing efficiency (~80%) in monolayer cultures and moderate efficiency (~12%) in HEOs. Unlike monolayer cultures, where EGFP-positive cells were enriched by FACS for downstream analysis, organoids were analyzed as bulk populations, and the coexistence of edited and non-edited cells during organoid expansion may further lower th.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
KAPA HiFi hotstart Master Mix	roche	kk2631
1M HEPES	Dalian Meilun Biotechnology	MA0036
A83-01	Selleck Chemicals	S7692
accutase	STEMCELL Technologies	07920
Advanced DMEM/F12	Thermo Fisher Scientific	2842961
Agarose	Thermo Fisher Scientific	75510019
B27 minus vitamin A	Thermo Fisher Scientific	12587010
BsmBI-v2	New England Biolabs	R0739S
CellTiter-Meiluncell Luminescent Cell Viability Assay reagent	Dalian Meilun Biotechnology	PWL111
CHIR-99021	Sigma-Aldrich	SML1046
collagen type I solution from rat	Sigma-Aldrich	C3867-1VL
DMEM	Thermo Fisher Scientific	6125120
DMEM	Thermo Fisher Scientific	11885084	low glucose
DPBS	Thermo Fisher Scientific	6125527
EDTA	Dalian Meilun Biotechnology	MB5737
Ethanol	China National Pharmaceutical Group Corporation	10009218
Fetal bovine serum	Dalian Meilun Biotechnology	F0226F
Gibson Assembly Master Mix	ABclonal Biotechnology	RM20523
Glacial acetic acid solution	China National Pharmaceutical Group Corporation	10000208
GlutaMAX	Thermo Fisher Scientific	2277245
HBSS	Thermo Fisher Scientific	14175095	no calcium, magnesium, phenol red
HBSS	Dalian Meilun Biotechnology	MA0034	with calcium, magnesium, phenol red
Hepatocyte Culture Medium (HCM)	Lonza	CC3198
Human [Leu15]-gastrin I	Tocris Bioscience	10047-33-3
Liberase	Sigma-Aldrich	5401119001
MagMAX Pure Bind Beads	Thermo Fisher Scientific	A58523
Matrigel	Corning	354230
N-acetyl-L-cysteine	Sigma-Aldrich	A9165
Nicotinamide	Sigma-Aldrich	N0636
Opti-MEM	Thermo Fisher Scientific	41116155	serum-free transfection medium
PCR purification kit	zymo research	D4014
Penicillin-streptomycin	Dalian Meilun Biotechnology	MA0110
Percoll	cytiva	10313229
Plasmid DNA purification kit	Nanjing Vazyme Biotech	DC201-01
pMD2.G	Addgene	12259	VSV-G envelope expressing plasmid
Polybrene	Sigma-Aldrich	TR-1003-G
Polyethyleneimine	Yeasen Biotech	40816ES01
psPAX2	Addgene	12260	lentiviral packaging plasmid
Puromycin	Yeasen Biotech	60209ES10
Recombinant Human EGF	PeproTech	AF-100-15
Recombinant Human FGF-10	Peprotech	100-26-100
Recombinant Human FGF-7	Peprotech	96-100-19-50
Recombinant Human HGF	PeproTech	96-100-39-50
RoomTemp Sample Lysis Kit	Nanjing Vazyme Biotech	P073-01
Trypan blue	Dalian Meilun Biotechnology	MA0130
Y-27632	STEMCELL Technologies	72304
Zoletil 50	virbac Corporate	/	tiletamine–zolazepam anesthetic mixture
Consumables
0.22 μm filter	Merck Millipore	SLGPR33RB
0.45 μm low-protein-binding filter	Merck Millipore	SLHPR33RB
1.5 mL centrifuge	axygen	MCT-150-C
10 cm Culture Dish	NEST Biotechnology	704001
100,000 NMWL centrifugal ultrafilters	Merck Millipore	UFC9100096
24 well plate	NEST Biotechnology	702001
50 mL centrifuge tube	NEST Biotechnology	602052
70 µm cell strainer	Corning Incorporated	431751
Cel lifter	Corning Incorporated	CLS3008
Equipment
Automated cell counter Countess3	Thermo Fisher Scientific	A50302
Benchtop refrigerated centrifuge	Thermo Fisher Scientific	ST1R plus
Biological safety cabinet	SuzhouAntaiAirtech	BSC-1304IIA2
CO2 incubator	Thermo Fisher Scientific	I160
Fluorescence activating cell sort (FACS)	BD bioscience	BD influx
Horizontal electrophoresis system	biorad
Inverted fluorescence microscope	olympus	IX71
Multi-channel peristaltic pump	longer	LEAD15-88
Nanodrop	Hangzhou Allsheng instruments	nano-300
PCR Thermal cycler	biorad	S1000 Cycler
Qubit 3.0	Thermo Fisher Scientific	Q33216
Ultra-low temperature (−80 °C) freezer	Thermo Fisher Scientific	8950086V
Software
CRISPResso2	/	https://crispresso2.pinellolab.org/submission	Analysis of genome editing outcomes from deep sequencing data
Inference of CRISPR Edits (ICE) algorithm	/	https://ice.editco.bio/#/	CRISPR editing analysis algorithm for inference of indels