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Page 120 Ghaseminejad et al. J Transl Genet Genom 2022;6:111-25 https://dx.doi.org/10.20517/jtgg.2021.49

two-way ANOVA) in Sg5-treated animals (n = 5) compared to the untreated adRP animals (n = 8)
[Figure 4C]. Our results indicate long-term improvement in retinal function in the Sg5-treated group.

We further assessed these long-term outcomes by histology. Nine-month-old animals from untreated and
Sg5-treated groups were sacrificed and genotyped. Eyes were dissected, embedded, sectioned, labeled, and
imaged as previously described to assess long-term effects of the Rho.LΔ11Δ1 mutation and gene editing
treatments. As shown in [Figure 4E], significant RD and loss of rods are observed in older Rho.LΔ11Δ1
animals. In agreement with the functional recovery seen in our ERG analysis, RD was prevented in Sg5-
treated animals, again indicating long-lasting benefits of treatment.

CRISPR-mediated HDR as a treatment approach for adRP
Although knocking out the Rho.LΔ11Δ1 allele by inducing NMD via indels introduced by NHEJ was
effective in preventing RD in our animal model, we expected the strategy to be successful only in the two-
thirds of photoreceptors that would carry frame-shifting mutations. In the remaining one-third of cases, in-
frame mutations would be neutral or aggravating, and this is likely reflected by the total rod opsin levels in
treated groups averaging about 2/3 of WT levels [Figure 2B and D]. Therefore, we attempted to utilize a
CRISPR-induced HDR repair mechanism to improve treatment outcomes, as HDR should produce error-
free repairs. We designed an experiment to compare the efficacy of our single-guide-based therapy to HDR-
based repair. We designed a 120-nucleotide single-stranded repair template that spanned the Sg5 cut site,
with the Rho.LΔ11Δ1 sequence restored to WT; recombination would revert the Rho.LΔ11Δ1 sequence to
WT and eliminate the Sg5 target site. In addition, we included a mutation that induces the amino acid
change M13F; this innocuous change generates a binding site for anti-mammalian rod opsin monoclonal
antibodies 2B2 and 514-18, allowing us to identify cells that have undergone recombination by
immunolabeling [20,22] . Finally, we included a silent mutation that introduces an ApoI restriction site
[Supplementary Material 2].

To test this strategy, we injected embryos from a WT male and a WT/Rho.LΔ11Δ1 female with Cas9 and Sg5
alone to induce the NHEJ, or Cas9 and a combination of Sg5 and ssDNA template to induce HDR and
compared them to untreated controls. The amount of ssDNA template injected (200 pg per embryo) was
separately determined to be the maximum amount that could be injected without high levels of toxicity
[Supplementary Material 4]. At 14 dpf, the animals were sacrificed, and genomic DNA samples were
collected for PCR analysis and Sanger sequencing. One eye was solubilized for dot blot assays, while the
other was fixed for confocal microscopy.

Similar to our findings in the previous experiment, both rod opsin dot blot and histology were consistent
with RD in untreated heterozygous WT/Rho.LΔ11Δ1 animals that were prevented by the Sg5 treatment
(Figure 5A; n = 32 per group; P = 0.017 by Kruskal-Wallis test). Hence, the results of the previous
experiment were replicated. Significant RD was also prevented in the group of animals treated with Sg5 in
combination with the ssDNA repair template [Figure 5A and B]; n = 32 per group; P = 0.027 by Dunn’s
multiple comparisons. There was no statistically significant difference between the Sg5 and Sg5 + ssDNA
treatments. The genotypes of a subset of animals were identified via Sanger sequencing (Figure 5A, colored
data points). As anticipated, we found higher levels of rod opsin in WT animals compared to mutants in
both treated and untreated groups. Similar to the experiment shown in [Figure 2], we observed evidence of
mosaic editing by Sg5 in the sequencing traces, and no evidence of HDR repair.

HDR inefficiently repaired the Rho.LΔ11Δ1 allele
In order to assess the efficiency of HDR in the 36 animals treated with Sg5 + ssDNA, three different assays
were carried out: (1) Rho.L exon 1 was amplified by PCR from each animal, and restriction analysis was