Rapid multiplex liver gene-editing in mice using adeno-associated virus 8 or lipid nanoparticles

Somatic liver knockout (SLiK) is a method developed to rapidly generate liver-specific knockout of one or several genes. However, the original protocol relies on hydrodynamic tail vein injection (HTVI), a procedure associated with low transfection efficiency and animal discomfort due to cardiac stress. To address these challenges, we evaluated whether HTVI could be adapted to adeno-associated virus serotype 8 (AAV8) or lipid nanoparticles (LNPs). In addition, we sought to expand SLiK functionality by incorporating transgene overexpression together with gene knockout. Two AAV8 vectors were co-injected into Fah ^-/- /spCas9 ^Tg mice, each carrying one gRNA targeting Hpd and a second gRNA targeting gene of interest (GOI), together with a cytomegalovirus (CMV)-driven transgene cassette. This design enables dual knockout with concurrent overexpression in the same hepatocyte. LNPs were tested as an alternative non-viral method to deliver individual gRNA. Both AAV8- and LNP-mediated approach achieved near-complete Hpd inactivation within 2 months, without significant adverse effects. Using AAV8, we generated Cyp2c70 knockout mice that recapitulated the known bile-acid phenotype. We also established mice models with either humanized NTCP (SLC10A1) or AGXT by deleting the murine genes and overexpressing their human ortholog. Our platform offers a versatile strategy for rapid, multiplex hepatocyte gene editing for broader scientific community.