Generation and Analysis of Xenopus laevis Models of Retinal Degeneration Using CRISPR/Cas9

Feehan, Joanna M.; Stanar, Paloma; Tam, Beatrice M.; Chiu, Colette N.; Moritz, Orson L.

doi:10.1007/978-1-4939-8669-9_14

Cited by 9 publications

(9 citation statements)

References 21 publications

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“…Further characteristics such as the size, external development of the embryos, type of breed, and the large number of progeny, make Xenopus a suitable animal model [121,122]. There are two main species that are used in research, namely X. laevis (allotetraploid genome) and X. tropicalis (diploid genome) [122,123], and it is the former, the one that has been used to model retinal dystrophies, with a special focus on the rhodopsin gene. Knock-out models have been generated by the microinjection of Cas9 mRNA and sgRNAs into single cell embryos [83].…”

Section: Xenopusmentioning

confidence: 99%

Application of CRISPR Tools for Variant Interpretation and Disease Modeling in Inherited Retinal Dystrophies

Fuster-García

García‐Bohórquez

Rodríguez-Muñoz

et al. 2020

Genes

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Inherited retinal dystrophies are an assorted group of rare diseases that collectively account for the major cause of visual impairment of genetic origin worldwide. Besides clinically, these vision loss disorders present a high genetic and allelic heterogeneity. To date, over 250 genes have been associated to retinal dystrophies with reported causative variants of every nature (nonsense, missense, frameshift, splice-site, large rearrangements, and so forth). Except for a fistful of mutations, most of them are private and affect one or few families, making it a challenge to ratify the newly identified candidate genes or the pathogenicity of dubious variants in disease-associated loci. A recurrent option involves altering the gene in in vitro or in vivo systems to contrast the resulting phenotype and molecular imprint. To validate specific mutations, the process must rely on simulating the precise genetic change, which, until recently, proved to be a difficult endeavor. The rise of the CRISPR/Cas9 technology and its adaptation for genetic engineering now offers a resourceful suite of tools to alleviate the process of functional studies. Here we review the implementation of these RNA-programmable Cas9 nucleases in culture-based and animal models to elucidate the role of novel genes and variants in retinal dystrophies.

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Section: Xenopusmentioning

confidence: 99%

Application of CRISPR Tools for Variant Interpretation and Disease Modeling in Inherited Retinal Dystrophies

Fuster-García

García‐Bohórquez

Rodríguez-Muñoz

et al. 2020

Genes

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“…In X. tropicalis, Nakayama and colleagues laid the foundation and set out a simple CRISPR pipeline and use of mutations in the tyrosinase gene to generate albinism phenotypes, targeting the start codon, leading to frameshift mutation and KO ( Nakayama et al., 2013 ). CRISPR can be used to analyse gene function, and to replicate human disease mutations to generate mosaic targeted mutant F0’s and lines in Xenopus embryos ( Feehan et al., 2019 ; Macken et al., 2021 ; Naert et al., 2017 , 2020 ; Naert and Vleminckx, 2018 ; Nakayama et al., 2013 ).…”

Section: Introductionmentioning

confidence: 99%

An efficient miRNA knockout approach using CRISPR-Cas9 in Xenopus

Godden

Antonaci

Ward

et al. 2022

Developmental Biology

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“…Therefore, a gene-based treatment strategy for autosomal dominant disease involving genes such as RHO that do not have an associated haploinsufficiency disorder is to develop allele-specific therapeutics that knock out the mutant allele, leaving the endogenous WT allele unaffected [18] . Another potential solution is to replace the mutant gene with an exogenous WT copy.…”

Section: Introductionmentioning

confidence: 99%

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

Ghaseminejad¹,

Tam²,

Chiu³

et al. 2022

J Transl Genet Genom

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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.

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Generation and Analysis of Xenopus laevis Models of Retinal Degeneration Using CRISPR/Cas9

Cited by 9 publications

References 21 publications

Application of CRISPR Tools for Variant Interpretation and Disease Modeling in Inherited Retinal Dystrophies

Application of CRISPR Tools for Variant Interpretation and Disease Modeling in Inherited Retinal Dystrophies

An efficient miRNA knockout approach using CRISPR-Cas9 in Xenopus

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

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