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.

Keywords

Rhodopsin / autosomal dominant retinitis pigmentosa / CRISPR/Cas9 / gene editing / gene therapy

References

[1] Hamel C.Retinitis pigmentosa.Orphanet J Rare Dis2006;1:40 PMCID:PMC1621055

[2] Retinitis pigmentosa. Fighting Blindness Canada (FBC). Available from: https://www.fightingblindness.ca/eye-diseases-pathways/retinitis-pigmentosa/ [Last accessed on 12 Jan 2022]

[3] Verbakel SK,Boon CJF.Non-syndromic retinitis pigmentosa.Prog Retin Eye Res2018;66:157-86

[4] Daiger SP,Bowne SJ.Genes and mutations causing retinitis pigmentosa.Clin Genet2013;84:132-41 PMCID:PMC3856531

[5] Hartong DT,Dryja TP.Retinitis pigmentosa.Lancet2006;368:1795-809

[6] Athanasiou D,Bellingham J.The molecular and cellular basis of rhodopsin retinitis pigmentosa reveals potential strategies for therapy.Prog Retin Eye Res2018;62:1-23 PMCID:PMC5779616

[7] Rosenfeld PJ,McGee TL,Berson EL.A null mutation in the rhodopsin gene causes rod photoreceptor dysfunction and autosomal recessive retinitis pigmentosa.Nat Genet1992;1:209-13

[8] Hsu PD,Zhang F.Development and applications of CRISPR-Cas9 for genome engineering.Cell2014;157:1262-78 PMCID:PMC4343198

[9] Jinek M,Fonfara I,Doudna JA.A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science2012;337:816-21 PMCID:PMC6286148

[10] Wang D,Wang B.Optimized CRISPR guide RNA design for two high-fidelity Cas9 variants by deep learning.Nat Commun2019;10:4284 PMCID:PMC6753114

[11] Zaboikin M,Freter C.Non-homologous end joining and homology directed DNA repair frequency of double-stranded breaks introduced by genome editing reagents.PLoS One2017;12:e0169931 PMCID:PMC5241150

[12] Feehan JM,Stanar P,Ahmed SN.Modeling dominant and recessive forms of retinitis pigmentosa by editing three rhodopsin-encoding genes in Xenopus laevis using Crispr/Cas9.Sci Rep2017;7:6920 PMCID:PMC5537283

[13] Zelinka CP,Fadool JM.Targeted disruption of the endogenous zebrafish rhodopsin locus as models of rapid rod photoreceptor degeneration.Mol Vis2018;24:587-602 PMCID:PMC6128699

[14] Ferreira MG.Two modes of DNA double-strand break repair are reciprocally regulated through the fission yeast cell cycle.Genes Dev2004;18:2249-54 PMCID:PMC517518

[15] Arnoult N,Ma J.Regulation of DNA repair pathway choice in S and G2 phases by the NHEJ inhibitor CYREN.Nature2017;549:548-52 PMCID:PMC5624508

[16] Lem J,Calvert PD.Morphological, physiological, and biochemical changes in rhodopsin knockout mice.Proc Natl Acad Sci U S A1999;96:736-41 PMCID:PMC15206

[17] Kartasasmita A,Iskandar E,Fujimaki T.A novel nonsense mutation in rhodopsin gene in two Indonesian families with autosomal recessive retinitis pigmentosa.Ophthalmic Genet2011;32:57-63

[18] Meng D,Tsang SH.Therapy in rhodopsin-mediated autosomal dominant retinitis pigmentosa.Mol Ther2020;28:2139-49 PMCID:PMC7545001

[19] Feehan JM,Tam BM,Moritz OL.Generation and analysis of Xenopus laevis models of retinal degeneration using CRISPR/Cas9.Methods Mol Biol2019;1834:193-207

[20] Tam BM,Oprian DD.Mislocalized rhodopsin does not require activation to cause retinal degeneration and neurite outgrowth in Xenopus laevis.J Neurosci2006;26:203-9

[21] Adamus G,Arendt A,Mcdowell JH.Anti-rhodopsin monoclonal antibodies of defined specificity: Characterization and application.Vision Res1991;31:17-31

[22] Tam BM.Characterization of rhodopsin P23H-induced retinal degeneration in a Xenopus laevis model of retinitis pigmentosa.Invest Ophthalmol Vis Sci2006;47:3234-41

[23] Tam BM,Kaushal S,Moritz OL.Photoactivation-induced instability of rhodopsin mutants T4K and T17M in rod outer segments underlies retinal degeneration in X. laevis transgenic models of retinitis pigmentosa.J Neurosci2014;34:13336-48 PMCID:PMC4180472

[24] Hicks D.Differential immunogold-dextran labeling of bovine and frog rod and cone cells using monoclonal antibodies against bovine rhodopsin.Exp Eye Res1986;42:55-71

[25] BioTechniques - a simple way to treat PCR products prior to sequencing using ExoSAP-IT®. Available from: http://www.biotechniques.com/BiotechniquesJournal/2008/May/A-Simple-Way-to-Treat-PCR-Products-Prior-to-Sequencing-Using-ExoSAP-IT/biotechniques-45295.html [Last accessed on 12 Jan 2022]

[26] Bocchero U,Chiu CN,Moritz OL.Electrophysiological changes during early steps of retinitis pigmentosa.Invest Ophthalmol Vis Sci2019;60:933-43

[27] Karimi K,Lotay VS.Xenbase: a genomic, epigenomic and transcriptomic model organism database.Nucleic Acids Res2018;46:D861-8 PMCID:PMC5753396

[28] Wen RH,Tam B.Autophagy in Xenopus laevis rod photoreceptors is independently regulated by phototransduction and misfolded RHO^P23H.Autophagy2019;15:1970-89 PMCID:PMC6844500

[29] Tam BM.Dark rearing rescues P23H rhodopsin-induced retinal degeneration in a transgenic Xenopus laevis model of retinitis pigmentosa: a chromophore-dependent mechanism characterized by production of N-terminally truncated mutant rhodopsin.J Neurosci2007;27:9043-53 PMCID:PMC6672211

[30] Diakatou M,Erkilic N,Meunier I.Allele-specific knockout by CRISPR/Cas to treat autosomal dominant retinitis pigmentosa caused by the G56R mutation in NR2E3.Int J Mol Sci2021;22:2607 PMCID:PMC7961898

[31] Latella MC,Cocchiarella F.In vivo editing of the human mutant rhodopsin gene by electroporation of plasmid-based CRISPR/Cas9 in the mouse retina.Mol Ther Nucleic Acids2016;5:e389 PMCID:PMC5155324

[32] Maruyama T,Truttmann MC,Ingram JR.Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining.Nat Biotechnol2015;33:538-42 PMCID:PMC4618510

[33] Lin S,Alla RK.Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery.Elife2014;3:e04766 PMCID:PMC4383097

[34] Vartak SV.Inhibition of nonhomologous end joining to increase the specificity of CRISPR/Cas9 genome editing.FEBS J2015;282:4289-94

[35] Srivastava M,Sharma S.An inhibitor of nonhomologous end-joining abrogates double-strand break repair and impedes cancer progression.Cell2012;151:1474-87

[36] Tsai YT,Lee TT.Clustered regularly interspaced short palindromic repeats-based genome surgery for the treatment of autosomal dominant retinitis pigmentosa.Ophthalmology2018;125:1421-30 PMCID:PMC6109419

[37] Miller RF.Intracellular responses of the Müller (glial) cells of mudpuppy retina: their relation to b-wave of the electroretinogram.J Neurophysiol1970;33:323-41