## 3 Discussion
We conducted a preliminary iPSC-derived retinal organoid study to further investigate the effect of allele-specific knockout of G56R NR2E3 in a native environment and, more specifically, on the retinal differentiation potential. We show, for the first time, that G56R NR2E3 iPSCs can differentiate into retinal organoids. This is consistent with the fact that G56R is associated to progressive RP, which is not a congenital disorder. We also show that G56R-CRISPR iPSCs retain the potential for retinal differentiation. Although, a thorough comparison of the phenotypic differences between WT, G56R, and G56R-CRISPR organoids was beyond the scope of this study, we observed that G56R-CRISPR2 organoids expressed NR2E3 in a lamination that was indistinguishable from WT. This suggests that _NR2E3_ allele-specific ablation would mimic the situation of ESCS loss-of-function mutation carriers who are asymptomatic, and that clinical translation of our CRISPR/Cas-mediated knockout strategy would not have negative effects on the human retina. Along this line, a previous study reported the generation of iPSC-derived retinal organoids from homozygous carriers of _NR2E3_ mutations [49]. In non-laminated retinal organoids, the authors detected S cones in patient organoids. By contrast, no S cones were detected WT organoids at the same differentiation timepoint, which suggested that the homozygous _NR2E3_ organoids mimicked the ESCS phenotype. iPSC-derived retinal organoids from heterozygous carriers were not generated in this previous study. Thus, the future characterization of G56R-CRISPR organoids in comparison to WT, in addition to validating our therapeutic strategy, will also indirectly provide information on the phenotype of _NR2E3_ ESCS carriers. Furthermore, although the phenotype of _NR2E3_ carriers suggest that a single allele will be sufficient for correct photoreceptor function, we do not yet exclude the necessity of coupling mutant allele knockout to _NR2E3_ gene supplementation. The G56R and G56R-CRISPR organoids will also provide the opportunity to directly address this question in a pertinent setting. Nevertheless, allele-specific knockout approaches via CRISPR/Cas have been successfully used in vivo in the case of certain IRDs, thus we are relatively confident that this would also be the case for the _NR2E3_ approach described herein. The recurrent P23H mutation in rhodopsin was one of the first mutations to have been successfully targeted by such a strategy. Proof-of-concept studies in P23H mouse models demonstrated reduced expression of the mutant protein [50], as well as retinal preservation and improved retinal function [51; 52]. Another highly pertinent target is the recurrent c.2991+1655A>G variant in intron 26 of the gene _CEP290_[53], which was associated with autosomal recessive Leber congenital amaurosis. Allele-specific knockout of the intronic variant restored WT _CEP290_ splicing in animal models [54; 55], and it has led to the first-in-humans Phase 1/2 clinical trial for CRISPR/Cas genome editing of a genetic disorder (NCT03872479). This suggests that clinical translation of a similar strategy for NR2E3 G56R may not be a pipedream. Allele-specific knockdown (as opposed to knockout) can also be accomplished using antisense oligonucleotide (AON)-based approaches, which have the advantage of being considered to be safer, as the host DNA is not targeted, but also the drawback of requiring repeat administration due to AON turnover. The _CEP290_ intron 26 variant has served as the target for AON-based proof-of-concept studies [56; 57], which have also led to Phase 1/2 clinical trials (NCT03140969). Along this line, an AON approach that was designed to specifically target the G56R variant was previously reported for _NR2E3_[58]. Encouragingly, a reduction in both G56R _NR2E3_ mRNA and protein levels was achieved. However, a reduction in WT mRNA and protein levels was also observed, indicating a lack of target specificity for the mutant allele.