## 3 Discussion  
The G56R mutation in NR2E3 is the second most common mutation causing adRP, a disorder for which there is currently no cure [3]. Genome editing offers a host of therapeutic options for IRDs in terms of gene and cell therapy [37], but the feasibility of clinical translation may be variable. For example, gene correction requires HDR and, thus, may not reach therapeutic efficiency for gene therapy, due to the post-mitotic nature of photoreceptors, but holds promise for cell therapy. By contrast, a HDR-independent strategy that involves specific knockout of a mutant allele could be a potentially efficient gene therapy approach. This would be particularly pertinent in the case of G56R, as this mutation exclusively causes all \(NR2E3\)-associated adRP forms [4]. Therefore, a single therapeutic product could potentially treat all patients, which renders it economically attractive. Here, for the first time, we developed a CRISPR/Cas9-mediated allele knockout strategy to treat \(NR2E3\)-associated adRP. The efficiency and specificity of this approach suggests that it could be a promising future treatment for \(NR2E3\) patients. Of the two allele-specific knockout systems that we designed, the most optimal was the one that was made up of the gRNA1 molecule spanning the G56R mutational site and adjacent to a NGG PAM sequence that can be recognized by eSpCas9. The combination of the allele specificity of the gRNA and the enhanced specificity feature of eSpCas9 resulted in specific targeting of the mutant allele in 75% of the surviving iPSC clones; in the remaining 25%, no genome editing events took place. Importantly, the wild type allele was never targeted in the surviving clones, and no off-target events were detected. The edited clones contained 1- or 2-bp indels within exon 2 of \(NR2E3\), which gave rise to a PTC. It has been  
Figure 6: Retinal differentiation potential following \(NR2E3\) allele knockout. Bright-field images of WT (**A**), G56R (**E**), and G56R-CRISPRR (**I**) iPSC-derived retinal organoids at 180 days of differentiation. IF analysis of NR2E3 expression (in green) in WT (**B**), G56R (**F**), and G56R-CRISPRR2 (**J**) retinal organoids; nuclei labelled in blue. NR2E3 expression is localized to the photoreceptor nuclei, which form an outer layer, corresponding to the outer nuclear layer (ONL), in the organoid. Colocalization studies of NR2E3 (in green) and OTX2 (in red) expression in WT (**C**), G56R (**G**) and G56R-CRISPRR2 (**K**) retinal organoids. NR2E3 is restricted to the rod nuclei whereas OTX2 also labels the more peripheral cone nuclei, as well as retinal cells situated on the inner side of the ONL. Colocalization studies of CRX (in green) and NRL (in red) expression in WT (**D**), G56R (**H**), and G56R-CRISPRR2 (**L**) retinal organoids. NRL expression is restricted to rod nuclei, whereas CRX also labels the cone nuclei situated on the outer rim of the organoids. established that NMD takes place when a PTC emerges \(\geq\)50-55 nt upstream of the last exon-exon junction [38,39]. In our case, the PTCs were located \(>\)800 nt upstream of the last exon 7 to exon 8 junction. Therefore, it is highly likely that the mRNA that is transcribed from the edited DNA would undergo NMD, before producing a mutant protein, which corresponds to an effective knockout. In this way, we can exclude the risk that the truncated protein further interferes with the WT NR2E3 allele. Nevertheless, it has been reported that, after CRISPR/Cas-mediated knockout, mRNAs can escape NMD. This can happen through different mechanisms, such as exon skipping, the use of alternative initiation codons, or conversion of mRNAs with a PTC into protein-coding molecules [40]. In addition, a truncated protein is sometimes detectable [41]. We overexpressed the G56R-CRISPR cDNA in an exogenous system wherein NMD would not occur in order to investigate what would happen at the protein level in case the mRNA escaped NMD [42].