Fig 2: The effects of wild-type rhodopsin and its mutants on cell survival and energy production. (A) The 661w cells were overexpressed with WT RHO, and the protein levels of RHO in cells were detected by immunoblotting at 24, 36, 48, and 60 hours after transfection. Next, cells were overexpressed with WT RHO and its missense mutants (P23H, R135L, and G188R), and RHO levels were detected by immunoblotting at 48 hours after transfection. (B) Cellular ATP levels and ATP/ADP ratios in each group were detected at 24, 36, 48, and 60 hours after transfection. (C) Cell apoptosis and necrosis were determined by flow cytometry with an Annexin-V FITC/PI Apoptosis Detection Kit at 24, 36, 48, and 60 hours after transfection. (D) The OCR and ECAR were analyzed using an Agilent Seahorse XFe24 Analyzer at 48 hours after transfection. (E) The protein levels of cyto c in the cytosolic fraction and c-cas3 were detected by immunoblotting at 48 hours after transfection. (F) The culture medium was changed to glucose-free DMEM at 24 hours after transfection, and the cellular energy status and cell apoptosis were determined after another 24 hours. *P < 0.05 compared with control group at the same time point, #P < 0.05 compared with the WT group at the same time point (n = 4).

Fig 3: Differentiation of Müller progenitor cells following short‐term culture w/w FGF2, Taurine, Retinoic acid, IGF1 (FTRI) differentiation complex. Only positive cells were shown to illustrate the pattern of gene expression before and after differentiation. Confocal images of fixed cells subjected to immunofluorescence staining against neural stem cell specific markers PAX6, NOTCH1, and ßIII TUBULIN (A, B, and C respectively), against the postmitotic neuronal marker Neurofilament (NEFM) (D), against the Müller glia specific marker GLUTAMINE SYNTHASE (GLUT SYN) (E), and against the photoreceptors specific markers RHODOPSIN (RHO) and RECOVERIN (REC) (F and G, respectively). Cells stained with secondary antibody only as controls. (anti‐rabbit IgG 555 made in goat, anti‐mouse IgG 647 made in goat and anti‐rabbit IgG 555 isotype control made in goat) see supplementary Figure 4. Hoechst 33342 (blue) was used as a counterstain. Scale bar = 50 mm unless otherwise indicated. (H) Reverse transcription‐polymerase reaction (RT‐PCR) products showing expression of postmitotic retinal neurons glutamine synthase (GLUL), PAX6, SOX2, NOTCH1, ßIII TUBULIN (TUBB3), 160 kDa Neurofilament (NEF3) and short wavelength Opsin (OPN1).

Fig 4: Hyperoxia has long-term effects on inner retinal neuron function. (A, B) Amplitudes of filtered oscillatory potentials (OPs), calculated from the recorded ERG responses under scotopic conditions at P23 (A) and seven weeks (B). Both at P23 and seven weeks the amplitudes of OPs are significantly reduced in the hyperoxia groups compared to the normoxia group irrespective of the genotype of the animals (n = 4–6). *P < 0.05, **P < 0.01, ***P < 0.001. P23 Controls are Dio2+/+ & Dio2+/−, adult controls are Dio2+/−. Data were analyzed using two-way ANOVA, and pairwise post-hoc comparisons were performed using Tukey's method. Error bars: ±SEM.

Fig 5: Schematic representation of allele-specific sgRNA design for RHO-T17M mutation.(A) and confirmation of the specificity achieved using SaCas9 protein complexed with an sgRNA to target the mutant RHO sequence and its corresponding WT sequence in vitro (B), the full-length amplicon was 678 bp, the two truncated amplicons were 445 bp and 233 bp, respectively. The mutation c.50C>T (p.T17M) is indicated in red. PAM sequences were marked in orange. Exons were indicated by closed boxes.