Fig. 3: PE system precisely corrected the PDE6B Y347X mutation in rd1 mice in vivo.
From: In vivo prime editing rescues photoreceptor degeneration in nonsense mutant retinitis pigmentosa
a Schematic diagram of the PE system delivered by dual-AAV. Cas9(H840A) was split at site 713, at which the N-terminus followed by the epegRNA and nsgRNA was packaged in one AAV, whereas the C-terminus linked to the RTΔRnH domain was packaged in the other AAV. A trans-splicing intein system was employed to reconstitute the two AAVs into the full-length PE. b Timeline of the in vivo AAV-PE treatment. One-week-old rd1 mice were subjected to subretinal injection of the dual-AAV system. Three weeks after injection, PE-treated rd1 mice were subjected to a series of functional assays for vision evaluation. Then, the PE-treated rd1 mice were sacrificed at five weeks of age, and their eyeballs were collected for molecular biology and morphological analyses. Untreated littermates and WT mice of the same age were used as negative and positive controls, respectively. Created in BioRender61. c The editing efficiency of the PE system in vivo. Representative data are presented as the mean ± SD, n = 6. One-way ANOVA with Tukey’s multiple comparisons test. *p < 0.05; ***p < 0.001. d Western blot analysis of PDE6B expression in mouse eyeball lysates after treatment at P35. Uncropped blots in Source Data. e Representative immunofluorescence image of the rod photoreceptor layer marked with rhodopsin (red) at P35. ONL, outer nuclear layer; INL, inner nuclear layer; OS, outer segment. Scale bar = 25 μm. f Quantification of OS thickness in WT, rd1, and PE-edited mice at P35. The means ± SDs are shown, n = 6. One-way ANOVA with Tukey’s multiple comparisons test was used for comparisons. **P < 0.01; ***p < 0.001. g Representative retinal section of WT, rd1, and PE-edited mice with H&E staining at P35. ONL, outer nuclear layer; INL, inner nuclear layer; and GCL, ganglion cell layer. Above scale bar = 100 μm. Below scale bar = 50 μm. h Quantification of ONL thickness in WT, rd1, and PE-edited mice at P35. One-way ANOVA with Tukey’s multiple comparisons test was used for comparisons. The means ± SDs are shown, n = 6. ***p < 0.001.
