Figure 3
From: Hemophilia A ameliorated in mice by CRISPR-based in vivo genome editing of human Factor VIII
Site-specific integration and expression of human BDD-F8 transgene into the mouse Alb locus. (A) Schematic diagrams showing the BDD-F8 transgene knock-in strategy. Adult C57BL/6 mice received a mixture of two AAV8 vectors via tail vein injection. Vector AAV8-SaCas9-sg1 expresses SaCas9 and sg1 gRNA targeting the Alb intron 13 region. Vector AAV8-BDD-F8 contains the BDD-F8 transgene cassette flanked by two sg1 gRNA recognition sequences (red boxes, also illustrated in Fig. 1A). Liver samples were collected for analyses 4 weeks after viral injection. (B) PCR showing viral infection in the mouse liver with vehicle only (Veh) or the two AAV8 vectors at a 1:5 ratio and total dose of 1.2 × 1012, 1.2 × 1011, or 1.2 × 1010 vg/kg, as indicated. M, DNA molecular weight marker. The full-length gels are shown in Supplementary Fig. 6,A. (C) Representative NGS data showing WT and mutated (indel) sequence patterns at the targeted Alb locus, and the percentage of each variant. (D) ddPCR analysis showing indel abundance (%) and BDD-F8 transgene knock-in event rates (KI%) at the targeted Alb locus. Each column represents Poisson means ± SE. (E) RT-PCR analyses of mouse liver cDNA in a mouse with a total AAV dose of 1.2 × 1012 vg/kg. Amplicons were produced using the specific primers F1/R1 (red arrows, A) and F2/R2 (blue arrows, A) that span the 5′-junction or 3′-junction, respectively, at the insertion site. Full-length gels are shown in Supplementary Fig. 6,B–D. (F) Relative levels of hybrid Alb/BDD-F8 and endogenous mouse Alb and F8 mRNAs in mouse livers. Data are means ± SE from 3–4 mice per group. (G) Representative immunofluorescence staining images, showing sporadic expression of human BDD-FVIII protein (red) in liver sections. Scale bars, 10 µm.
