Modeling ocular lens disease in <i>Xenopus</i>

Viet, Justine; Reboutier, David; Hardy, Serge; Lachke, Salil A.; Paillard, Luc; Gautier-Courteille, Carole

doi:10.1002/dvdy.147

Cited by 17 publications

(16 citation statements)

References 54 publications

(72 reference statements)

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“…In step 2, we examined the effects of varying the amount of Cas9 mRNA per embryo, as it has been shown that increasing the Cas9 mRNA amount to a certain level improves gene disruption efficiency in X. tropicalis (Blitz et al, 2013; Nakayama et al, 2013). Although previous studies have cultured injected embryos at various temperatures ranging from 12 to 22°C, there are no reports examining the effects of varying the culture temperature while keeping all other experimental conditions constant (Banach et al, 2017; Carr et al, 2021; Feehan et al, 2017; Kato et al, 2021; Smith et al, 2020; Tsujioka et al, 2017: Viet et al, 2020). Therefore, in step 3, we examined the effects of varying the embryo culture temperature post injection.…”

Section: Introductionmentioning

confidence: 99%

“…Clustered regulatory interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) has become a popular genome editing tool in Xenopus laevis (Aslan et al, 2017; Banach et al, 2017; Bharathan & Dickinson, 2019; Carr et al, 2021; Delay et al, 2018; Delay et al, 2019; Ezawa et al, 2021; Feehan et al, 2017; Jaffe et al, 2016; Kato et al, 2021; Kim et al, 2021; Ledford et al, 2017; Martin et al, 2020; Naert et al, 2020; Ochi et al, 2017; Pickett et al, 2017; Smith et al, 2020; Square et al, 2020; Tandon et al, 2017; Tsujioka et al, 2017; Viet et al, 2020; Wang et al, 2015; Wen et al, 2019; Wyatt et al, 2021). Importantly, most studies to date delivered the CRISPR reagents using a microinjection into fertilized eggs.…”

Section: Introductionmentioning

confidence: 99%

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Optimization of CRISPR/Cas9‐mediated gene disruption in Xenopus laevis using a phenotypic image analysis technique

et al. 2022

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The CRISPR/Cas9 method has become popular for gene disruption experiments in Xenopus laevis. However, the experimental conditions that influence the efficiency of CRISPR/Cas9 remain unclear. To that end, we developed an image analysis technique for the semi‐quantitative evaluation of the pigment phenotype resulting from the disruption of tyrosinase genes in X. laevis using a CRISPR/Cas9 approach, and then examined the effects of varying five experimental parameters (timing of the CRISPR reagent injection into developing embryos; amount of Cas9 mRNA in the injection reagent; total injection volume per embryo; number of injection sites per embryo; and the culture temperature of the injected embryos) on the gene disruption efficiency. The results of this systematic analysis suggest that the highest possible efficiency of target gene disruption can be achieved by injecting a total of 20 nL of the CRISPR reagent containing 1500 pg of Cas9 mRNA or 4 ng of Cas9 protein into two separate locations (10 nL each) of one‐cell stage embryos cultured at 22°C. This study also highlights the importance of balancing the experimental parameters for increasing gene disruption efficiency and provides valuable insights into the optimal conditions for applying the CRISPR/Cas9 system to new experimental organisms.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Optimization of CRISPR/Cas9‐mediated gene disruption in Xenopus laevis using a phenotypic image analysis technique

et al. 2022

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“…The main abnormities of the frog lens are shown in Figure 1. The picture of abnormities in the lenses of both eyes is often symmetrical; most likely, these are genotypic changes in the lens, and not acquired abnormities as a result of environmental contamination (Viet et al, 2020). Such changes account for about 12% of all abnormities.…”

Section: Resultsmentioning

confidence: 99%

Amphibian and fish eye lens used as biomarker of remote and chronic environmental contamination

Simakov

Nikiforov-Nikishin

et al. 2021

Ecol. Monten.

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The lens of vertebrates has a general structural plan and reacts similarly to various factors of anthropogenic impact. Rainbow trout (Oncorhynchus mykiss) and grass frog (Rana temporaria L.) are the most well-studied objects of aquatic toxicology in Russia, which react to the level of exposure to toxicants in the urbanized environment and retain the consequences of this exposure. The study aims to determine the possibility of using the lens of fish and amphibians as a biomarker of chronic and long-term background environmental contamination. The fundamental importance of the work lies in the fact that for the first time the mechanisms of the response of the epithelial cells of the eye lens and lens fibers to the spectrum of various toxicants entering the environment with industrial effluents are considered. In chronic experiments, the sensitivity of the lens of Oncorhynchus mykiss fingerlings to three toxicants (dibutyl sebacate-DBS, polyterpenes-PTP, and 3-aminopropyltriethoxysilane-AGM-9) in concentrations close to the normalized valuesin the environment was studied. On adult individuals of Rana temporaria, the appearance of abnormities in the lens was studied over a long period, depending on the total environmental contamination. To assess remote environmental contamination, studies were carried out on the constantly growing lens of the grass frog, which can preserve the resulting fiber abnormities throughout its life in the form of local opacities from the harmful environmental factors impacts. On the epithelium of the lens of fish, it was found that under the influence of certain pollutants, the mitotic index (MI) in the germinal area can change over a wide range. With the combined effect of toxicants, inhibition of mitosis is also observed. Using biomicroscopy of the eye, it was possible to show that frogs from the urbanized zone of Moscow have 1.5 times more genotypic and modification changes in the lens than their relatives taken from an ecologically clean zone of the Moscow region, Mozhaisky district in the water area of the Ozerninskoye reservoir. For the first time, the sensitivity of the lens of aquatic organisms to chronic effects under experimental and background contamination of the terrestrial and aquatic environment has been shown. Therefore, taking into account the uniformity of the structure of the eye lens of fish and amphibians, as well as in other vertebrates, the lens can be used as a biomarker for assessing chronic and remote environmental contamination. The research results can be applied for a comprehensive assessment of the action of anthropogenic factors.

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“…Not all of these have fully documented the underlying DNA sequence variation at the DSB site. Some did use next generation sequencing (NGS) [12][13][14] or Sanger sequencing of cloned genomic isolates from the embryos [14][15][16][17][18][19]. Others merely monitored the breakdown of Sanger traces from directly amplified DNA at the target site, combined with a deconvolution algorithm to predict likely repairs [20,21].…”

Section: Published Xenopus Crispr/cas9 Research -Can the Mosaicism That Results From Repair Of Cas9 Dsbs Be Eliminated?mentioning

confidence: 99%

How to increase precision in Xenopus gene editing and base editing experiments

Smith¹

2021

Preprint

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Modeling ocular lens disease in Xenopus

Cited by 17 publications

References 54 publications

Optimization of CRISPR/Cas9‐mediated gene disruption in Xenopus laevis using a phenotypic image analysis technique

Optimization of CRISPR/Cas9‐mediated gene disruption in Xenopus laevis using a phenotypic image analysis technique

Amphibian and fish eye lens used as biomarker of remote and chronic environmental contamination

How to increase precision in Xenopus gene editing and base editing experiments

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