Figure 5. In vivo genome editing with CjCas9 in the retina and retinal pigment epithelium.

(a) The CjCas9 target sequences in Vegfa and Hif1a/HIF1A genes. The PAM sequence and the sgRNA target sequence are shown in red and blue, respectively. (b) All-in-one AAV vector encoding CjCas9. (c) Indel frequencies at the Vegfa target site were analysed in RPE cells using deep sequencing at day 14, 28 and 42 post-intravitreal injection of AAV-CjCas9: Vegfa. Error bars indicate s.e.m. (n=4–5). One-way ANOVA and Tukey's post hoc tests, NS, not significant. (d) Representative confocal images of in vivo eGFP expression in RPE cells of AAV-CjCas9-injected mice 6 weeks after injection (n=6). eGFP was stained with anti-GFP antibody (green). Nuclei were counter-stained with DAPI (blue). Scale bar, 20 μm. (e–i) At day 42 post injection of AAV-CjCas9, indel frequencies and Vegfa protein levels were measured in retina and RPE cells using deep sequencing and ELISA, respectively. (e,f) Indel frequencies at the Rosa26, Vegfa and Hif1a target sites in the retina (e) and RPE cells (f). Error bars indicate s.e.m. (n=4 for AAV-uninjected control, n=5 for AAV-CjCas9). Student's t-tests, *P<0.05, ***P<0.001. (g,h) VEGFA levels measured by ELISA in the retina (g) and RPE cells (h), respectively. Error bars indicate s.e.m. (n=6–7). One-way ANOVA and Tukey's post hoc tests, *P<0.05, ***P<0.001. (i) Indel frequencies at in vitro cleavage sites identified by Digenome-seq. Genomic DNA isolated from RPE cells treated with AAV-CjCas9 at 6 weeks post injection was subjected to targeted deep sequencing. Mismatched nucleotides are shown in blue and PAM sequences in red. Red arrows indicate cleavage positions within the 22-bp target sequences.