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Ghaseminejad et al. J Transl Genet Genom 2022;6:111-25 https://dx.doi.org/10.20517/jtgg.2021.49 Page 123

similar treatment approach in human rhodopsin knock-in adRP mouse models using AAV injections.
Utilizing double sgRNAs in combination with Cas9 protein, they introduced DSBs upstream and
downstream from the rhodopsin gene’s start codon (363 bp apart), resulting in large inactivating deletions
in both alleles. Simultaneous with the elimination of the targeted gene, they enabled the expression of WT
rhodopsin through an exogenous cDNA . Our treatment approach differs in that one of the sgRNAs
[36]
designed to introduce the large inactivating deletion was designed to target only the mutant allele. Our
second non-allele-specific sgRNA was designed to introduce an edit upstream from the promoter regulatory
region, in a non-conserved region such that typical small indels would not be predicted to significantly
disrupt gene function. Therefore, our strategy did not require the delivery of an exogenous Rho cDNA.

Although the introduction of large deletions was only detected in a subset of animals (6 out of 11 tested),
this strategy could potentially be optimized and may also have significant utility in creating X. laevis
knockout models. One contributing factor to the efficiency of simultaneous breaks may be the physical
distance between the target sites. In our experiment, the sites were 1248 bp apart. Shorter or longer
distances may affect the efficiency of deletion. Another factor may be the timing of editing; unless both cuts
occur within a sufficiently narrow timeframe, two separate instances of NHEJ are more likely than a large
deletion.

Treatment with a single mutation-specific sgRNA conferred long-term protection against RD and loss of
retinal function, as late as 9 months of age, as assessed by histology and ERG. However, benefits were
primarily observed in the ERG a-wave, which is associated with the phototransduction functions of
photoreceptors. The b-wave is a reflection of communication between photoreceptors and bipolar and
Müller cells within the inner retina . Hence, we speculate that the reduction in a-wave amplitudes in our
[37]
adRP model is likely due to the reduced ability of rod photoreceptors to initiate phototransduction due to
missing or dysmorphic outer segments, while the relatively unaffected b-wave amplitudes indicate that a
large subset of viable rod photoreceptors remains at 9-months in WT/Rho.LΔ11Δ1 animals. Overall, our
results are consistent with functional impairment of rods in older animals that are prevented by gene
editing. Evaluation of long-term outcomes is another useful feature of this system, as X. laevis lifespan
routinely exceeds a decade in our hands.

CONCLUSION
Our results indicate that X. laevis can be used to rapidly model and compare therapeutic gene editing
strategies for adRP. From initial experiments in the system, we conclude that although more complex
editing strategies may be theoretically superior to simpler strategies, ultimately, they will lack superiority if
their complexity reduces efficiency. Therapies that require multiple linked events to occur in each cell (such
as two adjacent cuts within a narrow time frame, or a cut followed by recombination, or transfection with
two viruses) will require increased efficiencies for these independent events in order to surpass the efficacy
of theoretically inferior treatments requiring only a single step. Therefore, once the challenges of efficient
delivery of gene editing therapeutics are overcome, it should be possible to develop efficacious adRP
therapies based simply on NHEJ repair. There is likely merit to the idea of emphasizing the development of
less complex treatment strategies.

DECLARATIONS
Acknowledgments
Thanks to Keith Joung, Robert S. Molday, W. Clay Smith, and Dusanka Deretic for supplying plasmid and
antibody reagents.