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Page 116 Ghaseminejad et al. J Transl Genet Genom 2022;6:111-25 https://dx.doi.org/10.20517/jtgg.2021.49

Immunohistochemical labeling and confocal microscopy of contralateral eyes from genotyped tadpoles
further demonstrated that low rod opsin levels were due to an RD phenotype. In WT animals, healthy
retinas were observed with extended Rho-expressing rods and rod outer segments. In contrast,
WT/Rho.LΔ11Δ1 animals had significant RD apparent by confocal imaging, with disturbed and shortened
rod outer segments [Figure 1C].

sgRNA design
In order to target precise regions within Rho.L, we designed and generated sgRNAs as previously
[12]
described . We identified 20bp target sequences adjacent to PAM sites appropriate for CRISPR editing
[Supplementary Material 1]. We identified two target sites that incorporate the Rho.LΔ11Δ1 mutation
sequence in exon 1 (Sg5 and Sg6). In addition, we identified one site 1218 bp upstream of the start codon in
a less-conserved upstream promoter region of Rho.L that was not found in Rho.2.L or Rho.S (Sg2). In order
to guard against off-target effects, we confirmed that the Sg2, Sg5, and Sg6 target sites were unique by using
[27]
nblast to scan the X. laevis genome sequence available online at Xenbase.org . Notably, the Sg5 recognition
sequence only differs from WT by a single nucleotide [Figure 1A and Supplementary Material 1].

Comparing single-guide and double-guide CRISPR-based treatments
In our first experiments of gene-editing therapy in WT/Rho.LΔ11Δ1 animals, we utilized the Rho.LΔ11Δ1-
targeting sgRNAs (Sg5 and Sg6) either alone or in combination with the Rho.L promoter-targeting sgRNA
(Sg2) to evaluate the utility of these therapeutic strategies and compare their efficacy. The sgRNAs, along
with Cas9 protein and mRNA encoding eGFP (as a tracer to confirm successful delivery of RNA) were
injected into single-cell X. laevis embryos derived from a WT/Rho.LΔ11Δ1 female and a WT male. After
36 h, non-fluorescent embryos were eliminated from the experiment. At 14 dpf, the tadpoles were sacrificed,
genomic DNA was collected for PCR analysis, and the animals were genotyped by Sanger sequencing. One
eye was solubilized for a rod opsin dot blot assay, while the other was processed for confocal microscopy.

RD was prevented in animals treated with single and double-guide approaches
As shown in [Figure 2A and C], the single and double-guide treatments had minimal effects on WT
animals, with a mild toxic effect in the case of Sg6. However, treatment of WT/Rho.LΔ11Δ1 animals with
sgRNAs targeting the Rho.LΔ11Δ1 mutation resulted in significantly higher levels of rod opsin compared to
untreated animals [Figure 2B and D]. This effect was observed with both Sg5 and Sg6 guides [untreated (n =
9), Sg5-treated (n = 19), P = 0.000056; untreated (n = 11), Sg6-treated (n = 11), P = 0.0013 by Dunn’s test for
multiple comparisons]. However, average rod opsin levels were still approximately 1/3 lower than WT
animals. Combining the Sg5 and Sg6 guides with the Sg2 guide (targeting the upstream promoter) also
resulted in higher levels of rod opsin compared to the untreated WT/Rho.LΔ11Δ1 group, but was not
superior to the single-guide approach [untreated (n = 9), Sg2+5-treated (n = 10), P = 0.018; untreated (n =
11), Sg2+6-treated (n = 7), P = 0.032 by Dunn’s test for multiple comparisons]. There was no significant
difference between treatments with a single guide vs. two guides.

As shown in [Figure 2E], the Rho.LΔ11Δ1 allele results in significant RD and loss of rods. RD was prevented
in animals treated with either the Sg5 or Sg6 guides. Single-guide treatment of WT embryos with Sg5 and
Sg6 did not have detrimental effects, confirming the specificity of the guides for the mutant allele, as NHEJ
[12]
of the WT allele would be expected to cause RD .

RD was also significantly prevented in animals simultaneously treated with two sgRNAs, in both Sg2+Sg5
and Sg2+Sg6 groups. Overall, although the rod opsin levels were increased [Figure 2B and D] and RD was
prevented in the group treated with two guides [Figure 2E], this treatment approach was not statistically
superior to the single-guide approach.