## 2 Results
### Validating Allele-Specific Knockout of Mutant G56R NR2E3 at the Protein Level  
We tested _NR2E3_ expression levels in iPSCs by qPCR to validate our allele-specific knockout strategy at the mRNA level. However, this was inconclusive, as, firstly, _NR2E3_ was expressed at very low levels (>30 cycles) and, secondly, we were not able to differentiate between the expression of the WT, G56R, and G56R-CRISPRR2 and -3 alleles. Accordingly, we moved on to directly evaluate the effect of our genome-editing strategy at the protein level while using a cell-based overexpression assay. We first amplified WT _NR2E3_ cDNA from a commercial pool of retinal cDNA by PCR and then cloned the amplicon into the pGEM-T Easy vector system. Using this construct as a base, we performed site-directed mutagenesis and generated two additional plasmids, one containing the G56R mutation and the other, the G56R-CRISPRR2 frameshift mutation (we did not introduce the G56R-CRISPRR3 and -4 frameshift mutation, as the predicted protein truncation was identical to G56R-CRISPRR2). Subsequently, we sub-cloned the three _NR2E3_ variants (WT, G56R and G56R-CRISPRR2) into the pcDNA3 expression plasmid. The constructs were then separately transfected into HEK293 and COS7 cell lines and we performed western blot and IF studies 48 h post-transfection. We tested two antibodies in parallel to detect NR2E3 expression by western blot analysis: a mouse monoclonal antibody (clone H7223) directed to amino acids 2-45 of NR2E3 and a rabbit polyclonal directed to the whole NR2E3 protein. Immunohistochemistry of the cells that were transfected with the WT and G56R plasmids detected a full-length 45-kDa NR2E3 protein in both HEK293 (Figure 5A,B) and COS7 cell lines, as expected. By contrast, immunoblotting of the G56R-CRISPRR2-expressing cells only revealed a small protein that corresponded to the predicted \(\sim\)16 kDa size of the truncated NR2E3 (Figure 5A; 55-kDa tubulin band served as a loading control). Interestingly, only the mouse monoclonal antibody detected the 16-kDa band, whose epitope remains intact in G56R-CRISPRR2, and not by the polyclonal antibody (Figure 5B; a 42-kDa actin band served as a loading control), which suggested that the polyclonal antibody has a higher affinity for the C-terminal part of the protein that was lost in the G56R-CRISPRR2 clone. Following the western blot results, we only used the mouse monoclonal NR2E3 antibody for IF analyses to ensure that we would detect truncated NR2E3 expression. Following the transfection of HEK293 and COS7 (Figure 5C) cells with the three constructs, we detected protein expression in all cases, but the cellular localization differed strikingly between the three. WT NR2E3 showed a mixed perinuclear and nuclear localization (Figure 5C, left), whereas G56R NR2E3 preferentially showed a nuclear localization (Figure 5C, middle). We quantified three independent IF experiments to evaluate the different localization between WT and G56R. The analysis demonstrated that 89 \(\pm\) 1.9% of WT-transfected cells showed a perinuclear NR2E3 localization, as compared to 32 \(\pm\) 4.8% of G56R-transfected cells (Figure 5D). Conversely, 10.8 \(\pm\) 2% of WT-transfected cells showed a purely nuclear NR2E3 localization as compared to 68 \(\pm\) 4.8% of G56R-expressing cells (Figure 5E). These differences were statistically significant in both cases (_p_ < 0.05). Figure 5: Effect of mutant allele knockout on NR2E3 expression. Western blot analysis of HEK293 cells mock-transfected or transfected with the pCDNA3 constructs expressing WT, G56R or G56R-CRISPR2 NR2E3 and hybridized with **(A)** a mouse monoclonal anti-NR2E3 antibody (NR2E3 (m); in red) or **(B)** a rabbit polyclonal NR2E3 antibody (NR2E3 (r); in green). Both anti-NR2E3 antibodies detected a 45-kDa protein in WT and G56R-expressing cells. The mouse monoclonal anti-NR2E3 antibody detected a truncated 16-kDa protein in the G56R-CRISPR2-expressing cells **(A)**.