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Page 112 Ghaseminejad et al. J Transl Genet Genom 2022;6:111-25 https://dx.doi.org/10.20517/jtgg.2021.49

Conclusion: Our results demonstrate the utility of this new Xenopus laevis model for rapidly assessing and
comparing multiple gene-editing based treatment strategies. We conclude that it would be technically difficult to
improve on the simple single-guide based strategy, as strategies requiring multiple successive events (such as
cleavage followed by homology-directed repair) are likely to be less efficient.

Keywords: Rhodopsin, autosomal dominant retinitis pigmentosa, CRISPR/Cas9, gene editing, gene therapy

INTRODUCTION
Retinitis pigmentosa (RP) is a vision-threatening disorder characterized by loss of functional and viable
[1]
photoreceptors and retinal degeneration (RD) . RP affects 1 in 3500-4500 people worldwide, making it one
of the most common inherited retinal diseases . RP is rod-cone dystrophy, in which initial symptoms of
[2]
night blindness associated with rod degeneration progress to secondary cone death, resulting in the loss of
daylight peripheral vision, potentially leading to complete blindness .
[3]
Although most cases of RP are monogenic, thousands of different mutations in more than 50 genes underlie
this disorder . This genetic heterogeneity has made treatment development complex, and RP is untreatable
[4]
for more than a million affected individuals worldwide . In 30%-40% of cases, RP is inherited as an
[5]
autosomal dominant trait (adRP) . Notably, 25% of adRP cases stem from mutations in the rhodopsin gene
[5]
(RHO), making RHO mutations the most common cause of adRP. To date, more than 150 distinct
[6]
missense/nonsense RHO mutations have been associated with adRP . Mutations in RHO can also cause
recessive RP; in these cases, the mutations are typically null alleles .
[6,7]
CRISPR/Cas9 is a two-component gene editing system found in bacteria species that has been co-opted by
molecular biologists to permit a diverse range of genetic manipulations . The CRISPR/Cas9 complex is
[8]
made up of a DNA-cleaving enzyme called Cas9 and an RNA molecule that guides Cas9 to a specific target
site, referred to as single-guide RNA (sgRNA) . SgRNAs recognize unique ~20 nucleotide target sequences
[9]
flanked by a protospacer-adjacent motif (PAM) e.g., NGG in the case of S. pyogenes Cas9 . Cas9 introduces
[10]
double-strand breaks 3bp upstream from the PAM site. When the CRISPR/Cas9 system is employed in
vertebrate cells, the resulting DNA cleavage initiates either non-homologous end joining (NHEJ) or
homology-directed recombination (HDR) DNA repair pathways . NHEJ is an error-prone response that
[11]
results in insertions and deletions (indels) of short DNA sequences at the break site. Within RHO genes,
NHEJ can create frame-shifting indels that promote nonsense-mediated decay (NMD) of the mRNA,
generating loss-of-function mutations and a knockout (KO) phenotype [12,13] . However, in-frame indels in
RHO can cause photoreceptor degeneration via gain-of-function phenotypes such as rhodopsin misfolding
or instability [12,13] .

In contrast, the HDR pathway utilizes a homologous DNA sequence as a template for repairing double-
stranded break sites . In experimental settings, the homologous template sequence can be specifically
[14]
designed donor sequences of interest, allowing the introduction of novel DNA sequences. Hence, accurate
[15]
gene repair can be carried out utilizing the HDR pathway .
A previous study has shown that heterozygous Rho KO mice do not develop RD , although they have half
[16]
the normal complement of Rho, and their photoreceptor OS are shorter compared to wildtype (WT)
controls . Furthermore, parents of human patients with autosomal recessive RP caused by RHO null alleles
[16]
(i.e., heterozygous carriers) do not develop RP, although they have slightly reduced rod sensitivity [7,17] . Thus,
phenotypes associated with single RHO null alleles are significantly milder than the phenotypes associated