Figure 6: CjCas9 targeted to Vegfa or Hif1a reduces the area of laser-induced CNV in mice.
From: In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni
(a) At day 42 post injection of AAV-CjCas9, mice was treated with laser to induce choroidal neovascularization (CNV). One week after laser treatment, the CNV area was analysed. (b) In vivo expression of eGFP coexpressed with CjCas9 in laser-induced CNV. Representative confocal images of eGFP expression in the RPE of laser-induced CNV, 6 weeks after injection of AAV-CjCas9: Hif1a. eGFP was stained with anti-GFP antibody (green), CNV was stained with anti-IB4 antibody (red), and nuclei were counter-stained with DAPI (blue). Scale bar, 200 μm. Arrows indicate eGFP-expressing RPE cells. Scale bar, 100 μm (enlarged image). (c) Representative laser-induced CNV stained with isolectin B4 in the mouse eye injected with AAV-CjCas9 targeted to Rosa26, Vegfa or Hif1a. Scale bar, 200 μm. (d) The CNV area. Error bars indicate s.e.m. (n=17–18). One-way ANOVA and Tukey’s post hoc tests, *P<0.05; **P<0.01; ***P<0.001; NS, not significant. (e,f) At day 56 post AAV injection, full-field electroretinogram (ERG) was performed to evaluate cone function in mice. There was no significant decrease of b-wave of photopic response (e) and 30 Hz flicker response (f) in both AAV-CjCas9: Vegfa (n=8) or AAV-CjCas9: Hif1a (n=6) treated mice, compared to normal control mice (n=8). Error bars indicate s.e.m. (n=6–8). (g,h) Opsin-positive areas in the retina at day 42 post injection. (g) Representative images of opsin-positive areas in contact with RPE cells expressing HA-tagged CjCas9 in AAV-CjCas9: Rosa26, Vegfa or Hif1a injected mice compared with the AAV-uninjected negative control mice (no AAV). Opsin (red) and DAPI (blue). Scale bar, 20 μm. ONL, outer nuclear layer; IS, inner segment of photoreceptor cells; OS, outer segment of photoreceptor cells. See also Supplementary Fig. 6. (h) Relative opsin areas of the AAV-CjCas9-injected mice were normalized to that of the AAV-uninjected negative control mice. Error bars indicate s.e.m. (n=4). One-way ANOVA and Tukey’s post hoc tests, *P<0.05.
