Figure 2.

CNGB3 Targeting Efficiency Tests in Monkey Cone Photoreceptors Infected with AAVs

(A) Dissected retina showing strong DsRed signal, revealing the expression of the CRISPR-Cas9 system. (B) Statistics of indel reads and matched reads in deep sequencing show the targeting efficiency at the genome level. (C) Coimmunostaining of CNGB3 and M/S-opsin in the control retina. These proteins localized in the outer segments of the photoreceptors and showed strong overlapping signals. OS, outer segment; ONL, outer nuclear layer. The scale bar representing 25 μm is indicated at the bottom right of the figure. (D) Representative staining image of cone-specific photoreceptor antibodies, M/S-opsin and CNGB3, in the macular region, which shows the specificity of AAV9. AAV-CNGB3sgRNA-DsRed is indicated in pink. OS, outer segment; IS inner segment; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. The scale bar representing 50 μm is indicated at the bottom right of the figure. (E) Representative image showing CNGB3 knockout cones, as indicated by immunofluorescence. CNGB3 (green) and M/S-opsin (red) were used to survey the knockout efficiency in cones. Those cones are marked with yellow polygons. The scale bar representing 50 μm is indicated bottom right in the figure. (F) Statistics for (E). Each column represents the means of n = 2 animals. The p value was calculated with t tests (t = 7.071, df = 2) and is indicated in the figure. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; n.s, no significance.