Figure 3.

In vivo delivery of the CRISPR/Cas9 system into the rat HTI model via recombinant adenoviral vectors. A, left, schematic views of the recombinant adenoviral vector design and the strategy to repair the Fah gene in Fah^Δ10/Δ10 rats. The sgRNA target site is listed under the Fah^Δ10/Δ10 allele. The dashed line in the target site sequence represents the 10-bp deletion in the Fah^Δ10/Δ10 rat. The theoretically repaired sequence is listed under the HDnR allele. The red-dashed rectangle indicates the fully repaired Fah sequence. The PAM sequence is labeled in blue. Silent mutations are labeled in green. Right, an overview of the NTBC withdrawal process; the black lines indicate the days under NTBC treatment and the gray lines indicate NTBC withdrawal periods. PHT, partial hepatectomy. B, co-infection of the AdV–Cas9/AdV–Cas9n (red) and the AdV-HDR (GFP). Scale bar, 200 μm. C, indel frequencies caused by AdV–Cas9 or AdV–Cas9n in the rat liver on day 8. Data were obtained after sequencing 98 clones. D, immunohistochemistry staining of rat Fah protein in liver tissues from AdV–Cas9 and AdV–Cas9n groups on day 8. Fah^Δ10/Δ10 and WT rats served as negative and positive controls, respectively.