Subretinal injection of Cas9 RNPs targeting Vegfa reduces the area of laser-induced choroidal neovascularization (CNV) in a mouse model of age-related macular degeneration (AMD). (A) Mice with laser-induced CNV were treated with subretinal injection of the Vegfa-specific preassembled Cas9 RNP (Vegfa-RNP). After the retinal pigment epithelium (RPE) complex in the eye was flat-mounted, the CNV area was analyzed at day 7 post-injection. Genomic DNA isolated from the Cas9 RNP-injected area or from the opposite noninjected area (RNP-free area) was analyzed by deep sequencing. VEGFA ELISA was performed at day 3 post-injection. (B) Representative laser-induced CNV stained with isolectin B4 (IB4) in C57BL/6J mice injected with the Rosa26-specific Cas9 RNP (as a control) or the Vegfa-RNP. The yellow line demarcates the area of CNV. (C) The CNV area. Error bars indicate SEM (n = 15). Student's t-test: (***) P < 0.001. (D) VEGFA level in CNV. Error bars indicate SEM (n = 10). One-way ANOVA and Tukey post-hoc tests: (***) P < 0.001. (E) Indel frequencies at the Vegfa target site in the RPE cells. Error bars indicate SEM (n = 5). Student's t-test: (***) P < 0.001. (F) Indel frequencies at the Rosa26 target site in the RPE cells. Error bars indicate SEM (n = 5). Student's t-test: (***) P < 0.001.
