Approaching Perfection: New Developments in Zebrafish Genome Engineering

Shah, Arish N; Moens, Cecilia B.

doi:10.1016/j.devcel.2016.03.004

Cited by 6 publications

(7 citation statements)

References 10 publications

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“…Another major limitation for establishing a pure animal line with HDR mutation is the low efficiency of germline transmission (19,36). It has been reported that the germ materials in the zebrafish start to be detectable at 2-cell stage and become specialized at 4-cell stage.…”

Section: Crispr-mediated Point Mutation In the Zebrafish Discussionmentioning

confidence: 99%

“…The most challenging issue in creating single amino acid substitution is to differentiate HDR-induced point mutations from a large number of NHEJ-induced indel mutations, which are much more frequent in F1 generation. The methods reported so far such as HRMA and T7E1 assay are commonly used for screening mutations, but they are ineffective in distinguishing HDR-induced point mutations in F1 generation from random indel mutations (19,36).…”

Section: Establishment Of Screening Protocol For Differentiating Hdr-mentioning

confidence: 99%

“…In the past 20 years, zebrafish have become a popular model for reverse genetic analysis of vertebrate development and human diseases (15,19,(31)(32)(33)(34). Similar to other model organisms, zebrafish are amenable to genetic manipulation because of their small size, short life cycle, and high fecundity (33).…”

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confidence: 99%

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An efficient platform for generating somatic point mutations with germline transmission in the zebrafish by CRISPR/Cas9-mediated gene editing

Zhang¹,

Zhang²,

Ge³

2018

Journal of Biological Chemistry

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Homology-directed recombination (HDR)-mediated genome editing is a powerful approach for both basic functional study and disease modeling. Although some studies have reported HDR-mediated precise editing in nonrodent models, the efficiency of establishing pure mutant animal lines that carry specific amino acid substitutions remains low. Furthermore, because the efficiency of nonhomologous end joining (NHEJ)-induced insertion and deletion (indel) mutations is normally much higher than that of HDR-induced point mutations, it is often difficult to identify the latter in the background of indel mutations. Using zebrafish as the model organism and Y box-binding protein 1 (Ybx1/) as the model molecule, we have established an efficient platform for precise CRISPR/Cas9-mediated gene editing in somatic cells, yielding an efficiency of up to 74% embryos. Moreover, we established a procedure for screening germline transmission of point mutations out of indel mutations even when germline transmission efficiency was low (<2%). To further improve germline transmission of HDR-induced point mutations, we optimized several key factors that may affect HDR efficiency, including the type of DNA donor, suppression of NHEJ, stimulation of HDR pathways, and use of Cas9 protein instead of mRNA. The optimized combination of these factors significantly increased the efficiency of germline transmission of point mutation up to 25%. In summary, we have developed an efficient procedure for creating point mutations and differentiating mutant individuals from those carrying knockouts of entire genes.

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Section: Crispr-mediated Point Mutation In the Zebrafish Discussionmentioning

confidence: 99%

Section: Establishment Of Screening Protocol For Differentiating Hdr-mentioning

confidence: 99%

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An efficient platform for generating somatic point mutations with germline transmission in the zebrafish by CRISPR/Cas9-mediated gene editing

Zhang¹,

Zhang²,

Ge³

2018

Journal of Biological Chemistry

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“…To engineer zebrafish carrying the pbx4 p.A131V variant, we turned to the CRISPR-Cas9 system (Blackburn et al, 2013;Hwang et al, 2013;Hwang et al, 2015). Although there have been recent improvements, most studies report a low efficiency of introducing precise base changes using CRISPR-Cas9 in zebrafish (Hoshijima et al, 2016;Shah and Moens, 2016;Zhang et al, 2017;Zhang et al, 2018;and references therein), Therefore, to optimize our efforts, we first wanted to ensure that we were using efficient CRISPR-component reagents.…”

Section: Engineering Zebrafish Pbx4 Pa131v Variant Strainsmentioning

confidence: 99%

Functional testing of a humanPBX3variant in zebrafish reveals a potential modifier role in congenital heart defects

Farr

Imani

Pouv

et al. 2018

Preprint

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statementOur study provides a novel example of using genome editing in zebrafish to demonstrate how a human DNA sequence variant of unknown significance may contribute to the complex genetics of congenital heart defects. AbstractWhole-genome and whole-exome sequencing efforts are increasingly identifying candidate genetic variants associated with human disease. However, predicting and testing the pathogenicity of a genetic variant remains challenging. Genome editing allows for the rigorous functional testing of human genetic variants in animal models. Congenital heart defects (CHDs)

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“…Nowadays, the zebrafish is the most advanced aquatic species for the use of Cripsr-Cas9 in laboratories (Shah and Moens 2016). In this species, however, a challenge remains: efficient insertions of point mutations (for example, to mimic human missense mutations) are still generated at low rates (Renaud et al 2016).…”

Section: Introductionmentioning

confidence: 99%

Aquatic Model Organisms in Neurosciences: The Genome-Editing Revolution

Joly¹

2017

Research and Perspectives in Neurosciences

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The use of aquatic model organisms has been greatly diversified in laboratories. Zebrafish is the most advanced aquatic species for the use of Crispr-Cas9 in laboratories. Because of the simplicity and broad applicability of this later system, knock-out is now efficiently performed at medium scale. Forward genetics in zebrafish can now be performed by CRISPR-based F0 screening using high speed and high content phenotyping for example by confocal imaging. As zebrafish, marine model organisms have the prominent advantage to be transparent, all the more at young stages (embryos and larvae) or when fixed samples are cleared by novel methods. The Cripsr-Cas9 system is routinely used in the ascidian Ciona intestinalis. It also starts to be used in many other marine models, such as the medusa Clythia hemispherica. We provide at the end of this review a list of aquatic model species and some examples of questions on the origin of our nervous system that can be coped with these models, where the possibility to perform genome editing would constitute a major advance.

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Approaching Perfection: New Developments in Zebrafish Genome Engineering

Cited by 6 publications

References 10 publications

An efficient platform for generating somatic point mutations with germline transmission in the zebrafish by CRISPR/Cas9-mediated gene editing

An efficient platform for generating somatic point mutations with germline transmission in the zebrafish by CRISPR/Cas9-mediated gene editing

Functional testing of a humanPBX3variant in zebrafish reveals a potential modifier role in congenital heart defects

Aquatic Model Organisms in Neurosciences: The Genome-Editing Revolution

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