Fig. 1

High-level insertion editing of the liver at Alb by a double-cut donor after hydrodynamic injection. a Schematic of hydrodynamic injection. Plasmids encoding Cas9 and a sgRNA targeting the Alb stop codon (sgAlb), together with an HDR template (pDonor), were delivered to the liver by hydrodynamic tail vein injection. b Schematic of genome editing at the Alb stop codon. Knock-in of promoterless BDDF8 expression cassette at Alb was achieved by Cas9-sgAlb-mediated simultaneous cleavage of the genome and the double-cut donor pD-tdTomato-BDDF8-sg. The pD-sg template carries 600-bp homology arms, flanked by Cas9-sgAlb recognition sequences. Successful integration and transcription will produce three proteins (Alb, tdTomato, and BDDF8) as the result of E2A-mediated ribosomal skipping. c Knock-in efficiencies were determined by FACS analysis of tdTomato⁺ cells. Representative FACS diagrams are shown. d The double-cut donor considerably increases insertion efficiency in mouse liver cells after CRISPR-mediated dsDNA breakage. e Editing with the double-cut donor increases coagulant activity of F8. n = 12 mice for both conventional pD-tdTomato-BDDF8 and double-cut pD-tdTomato-BDDF8-sg donors. Omitting one or two editing components (n = 4 for each) served as negative controls. An unpaired t test with Welch’s correction was used for statistical analysis; ***P < 0.001. f A confocal liver section from edited mice shows expression of tdTomato in cells with hepatocyte morphology (representative of n = 5 mice). Scale bars are 100 μm