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Page 122 Ghaseminejad et al. J Transl Genet Genom 2022;6:111-25 https://dx.doi.org/10.20517/jtgg.2021.49

deletion in the Rho.L gene, was previously developed in our laboratory using the CRISPR/Cas9 gene editing
[12]
system . The in-frame deletion in the first exon of the X. laevis Rho.L gene results in a dominant RD
phenotype that is likely caused by defects in the biosynthesis of Rho protein, similar to previously described
mutations associated with adRP in humans (e.g., P23H) [28,29] . In support of this, we were unable to
convincingly detect the truncated mutant protein in the background of WT rod opsin by western blot using
the anti-C-terminal antibody mab11D5 (not shown). Successful gene editing therapies in this model have
similar requirements to those for human autosomal dominant disease, in that the mutant allele would either
[18]
need to be eliminated or restored . Thus, this model provides us with the means to rapidly survey a variety
of gene-editing treatment strategies for adRP. Due to the relative ease of gene editing in embryonic X. laevis,
the therapies were uncomplicated by issues such as delivery to the retina, or timing of delivery, both
currently the subject of extensive research. Therefore, our experiments model idealized scenarios in which
these issues have been solved.

Treating animals with a single sgRNA uniquely targeting the Rho.LΔ11Δ1 allele was overwhelmingly
successful. We expected NHEJ to result in out-of-frame mutations and nonsense-mediated decay in
approximately two-thirds of photoreceptors. Hence, loss-of-function mutations in the malfunctioning
alleles were projected to prevent the majority, but not all, of the RD. The treatment significantly prevented
RD in almost all animals, assessed by both dot blot for rod opsin and histology. Additionally, we were able
to demonstrate that a single-base discrepancy is sufficient for Cas9 to distinguish between the WT and
mutant alleles. This is particularly important for potential clinical use of gene editing strategies, as the
majority of adRP-associated RHO mutations reported in human patients are alterations of a single
nucleotide. Most recently, Diakatou et al. have published their work using a similar editing strategy to
[30]
treat G56R and NR2E3 adRP mutations in iPSCs. They designed unique sgRNAs to specifically target
mutant alleles with a single-base difference and successfully utilized CRISPR/Cas9 to introduce NHEJ-
[30]
mediated indels that KO the mutant allele . Latella et al. have also used a similar gene-editing approach
[31]
in mouse retinas carrying the human P23H rhodopsin gene, through subretinal electroporation of plasmid-
based Cas9. There are also limitations associated with NHEJ-based treatment for adRP. Although we have
shown that Cas9 is able to discriminate minor differences between disease and WT alleles, sgRNAs cannot
be designed for every known mutation due to the requirement for an adjacent PAM site. Our findings
emphasize that even an imperfect treatment strategy can be highly effective.

We also tested more complex editing strategies, designed to address the non-productive third of expected
in-frame edits. In attempting to restore the mutated allele to WT in our adRP model, we found that HDR is
inefficient in X. laevis using our described protocol. Although the reasons remain unclear, similar results
were also obtained by Feehan et al. in our laboratory. Here, we attempted to improve HDR efficiency
[12]
relative to previous attempts by using a single-stranded repair template, as opposed to the double-stranded
[12]
template used by Feehan et al. . However, HDR efficiency remained low relative to NHEJ. The incidence
[32]
and efficiency of HDR-mediated repair are also reported to be low in mammalian cells. Maruyama et al.
have reported HDR repair efficiencies ranging from 0.5% to 20%, compared to 20%-60% NHEJ-mediated
repair in mouse models. To improve HDR efficiency, Lin et al. have proposed a novel strategy to
[33]
synchronize cells into late S and G2 phases of the cell cycle, where HDR predominantly takes place.
Alternatively, other research groups have attempted to suppress NHEJ by targeting components such as
DNA ligase IV, forcing HDR to become the dominant repair pathway [34,35] .

We also used a gene inactivation strategy in which the transcription and translation start sites of the
mutated gene were removed by introducing DSBs upstream and downstream of the Rho.L start codon to
[36]
create large deletions. This also resulted in viable retinas without RD. Tsai et al. previously carried out a