Page 66 - Read Online

Ghaseminejad et al. J Transl Genet Genom 2022;6:111-25 Journal of Translational
DOI: 10.20517/jtgg.2021.49
Genetics and Genomics

Original Article Open Access

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

mutant Rhodopsin allele

1,2
1
1
1
Farhad Ghaseminejad , Beatrice M. Tam , Colette N. Chiu , Joanna M. Feehan , Orson L. Moritz 1
1
Department of Ophthalmology & Visual Sciences, University of British Columbia, UBC/VGH Eye Care Centre, Vancouver, BC
V5Z 3N9, Canada.
2
Current affiliation: The Sainsbury Laboratory, University of East Anglia, Norwich NR4 7UH, UK.
Correspondence to: Orson L. Moritz, Department of Ophthalmology & Visual Sciences, University of British Columbia,
UBC/VGH Eye Care Centre, 2550 Willow Street, Vancouver, BC V5Z 3N9, Canada. E-mail: olmoritz@mail.ubc.ca
How to cite this article: Ghaseminejad F, Tam BM, Chiu CN, Feehan JM, Moritz OL. Gene editing treatment strategies for
retinitis pigmentosa assessed in Xenopus laevis carrying a mutant Rhodopsin allele. J Transl Genet Genom 2022;6:111-25.
https://dx.doi.org/10.20517/jtgg.2021.49

Received: 28 Sep 2021 First Decision: 8 Nov 2021 Revised: 26 Nov 2021 Accepted: 9 Dec 2021 Published: 17 Feb 2022
Academic Editor: Bernhard H. F. Weber Copy Editor: Yue-Yue Zhang Production Editor: Yue-Yue Zhang

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.

© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0
International License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, sharing,
adaptation, distribution and reproduction in any medium or format, for any purpose, even commercially, as
long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and
indicate if changes were made.

www.jtggjournal.com