Gene editing treatment strategies for retinitis pigmentosa assessed in Xenopus laevis carrying a mutant Rhodopsin allele

Abstract

Aim: To examine the utility of gene editing therapies for retinitis pigmentosa using Xenopus laevis carrying a mutation in Rhodopsin. Methods: Xenopus laevis were genetically modified using CRISPR-Cas9 based methods and characterized by Sanger sequencing, dot blot, electroretinography, and confocal microscopy. Results: We identified genetically modified Xenopus laevis carrying a net 12 base pair deletion in the Rho.L gene. These animals have a retinal degeneration that is apparent by 14 days, with abnormal or missing rod outer segments, and a reduced electroretinogram signal. We prevented the majority of this retinal degeneration via a treatment strategy using a single sgRNA to neutralize the mutant allele via non-homologous end joining, yielding long-term improvements in histology and the electroretinogram. A second strategy using two sgRNAs to generate large deletions in the mutant allele was also successful, but did not significantly improve outcomes relative to the single-guide strategy as it was less efficient. We found limited evidence of success with a third strategy dependent on homology-directed repair; this treatment was also too inefficient to generate an outcome superior to the single-guide strategy. 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.