## 2 Results
### Designing a G56R-Specific Knockout Strategy  
The challenge of a G56R-specific knockout strategy was to design allele-specific gRNAs that would not target the wild type (WT) allele, which could only be achieved by gRNAs that spanned the mutational site. We used three online software for gRNA design: CCTOP ([[https://crispr.cos.uni-heidelberg.de](https://crispr.cos.uni-heidelberg.de)]([https://crispr.cos.uni-heidelberg.de](https://crispr.cos.uni-heidelberg.de))), CRISPR ([[http://crispr.tefor.net](http://crispr.tefor.net)]([http://crispr.tefor.net](http://crispr.tefor.net))), and MIT CRISPR online design tool ([[https://zlab.bio/guide-design-resources](https://zlab.bio/guide-design-resources)]([https://zlab.bio/guide-design-resources](https://zlab.bio/guide-design-resources)), no longer available), and identified two overlapping gRNAs that fit our criteria (Figure 2A). The first, gRNA1, had an NGG PAM sequence, which could be recognized by the SpCas9 as well as by its enhanced specificity variant, eSpCas9, which has reduced off-target effects [30]. The second gRNA, gRNA2, had an NGA PAM sequence, which could be recognized by the engineered VQR variant of SpCas9, which was created to increase the repertoire of genomic target sequences [31]. When designing gRNA2, we did not add a mismatched G in its 5\({}^{\prime}\), which has been described to enhance transcription that was driven by the U6 promoter in the expression plasmid [32], because the first nucleotide was already a G. Similarly, we did not include an extra G for gRNA1 that pairs with the eSpCas9, because we previously showed that this reduces its activity [33]. We next assessed the ability of the two gRNAs to selectively target the G56R allele. We cloned each gRNA into the corresponding Cas9 plasmid, which contained an enhanced  
Figure 1: Characterization of the G56R induced pluripotent stem cells (iPSC) line from the _NR2E3_-adRP patient. (**A**) Bright-field microscopy of a G56R iPSC colonies. (**B**) Sanger sequencing of exon 2 of _NR2E3_ showing a G at cDNA position 166 in the DNA of wild type (WT) iPSC and the G>A transition at c.166 in the G56R iPSC line. (**C**) RT-PCR detection of the reprogramming vectors in RNA from non-transduced patient fibroblasts (Fibro; negative control), fibroblasts transduced with the Sendai virus vectors (Fibro + SeV; positive control) and G56R iPSCs using primers specific for either the vector backbone (SeV) or for each reprogramming cassette: polycistronic _KLF4_, _OCT3/4_, and _SOX2_ (KOS), or monocistronic _KLF4_ and _MYC_. Primers for the housekeeping gene _GAPDH_ were used as a positive control for the PCR reaction. (**D**) Karyotype analysis of the G56R iPSC line showing normal chromosomal number and structure. (**E**) qPCR analysis of the expression of the host pluripotency genes _NANOG_ (left), _OCT3/4_ (middle), and _LIN28A_ (right) in cDNA from non-transduced patient fibroblasts (Fibro; negative control), fibroblasts transduced with the Sendai virus vectors (Fibro + SeV) and G56R iPSCs. The results are expressed as mean \(\pm\) SEM (\(n=3\)). (**F**) Immunofluorescence (IF) studies of the expression of endogenous NANOG (left), OCT3/4 (middle) and SOX2 (right) in G56R iPSCs; nuclei labelled in blue. (**G**) IF studies following embryoid body differentiation of G56R iPSCs for the expression of the germ layer markers Alpha Fetal Protein (AFP) (endoderm; left), Smooth Muscle Actin (SMA) (mesoderm; middle), and Nestin (ectoderm; right). green fluorescent protein (EGFP) tag, and nucleofected WT and G56R iPSCs. Two-days post-transfection, we isolated genomic DNA and performed a T7 endonuclease 1 (T7E1) assay. T7E1 digests DNA when there is mismatch between the two strands, as can occur if there is a frameshift mutation in one of the alleles. A positive digestion result is indicated by multiple bands after gel electrophoresis. The T7E1 assay was positive for both gRNAs in the patient iPSCs, indicating that at least one of the alleles had been targeted, as shown in (Figure 2B).