ArticleMolecular biotechnology2016
A Rapid and Cheap Methodology for CRISPR/Cas9 Zebrafish Mutant Screening.
Article in Molecular biotechnology, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 14 papers.
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Who cites it
14 citing papers in PubMed, 26 citations in OpenAlex.
- Brain Proteome and Behavioural Analysis in Wild Type, BDNFInternational journal of molecular sciences · 2022Article
- Review
- Loss of circadian rhythmicity iniScience · 2022Article
- Determining the Role of Maternally-Expressed Genes in Early Development with Maternal Crispants.Journal of visualized experiments : JoVE · 2021Article
- Physiological impact and comparison of mutant screening methods in piwil2 KO founder Nile tilapia produced by CRISPR/Cas9 system.Scientific reports · 2020Article
- MultiFRAGing: Rapid and Simultaneous Genotyping of Multiple Alleles in a Single Reaction.Scientific reports · 2020Article
- Modeling Neuronal Diseases in Zebrafish in the Era of CRISPR.Current neuropharmacology · 2020Review
- Genome sequencing and CRISPR/Cas9 gene editing of an early flowering Mini-Citrus (Fortunella hindsii).Plant biotechnology journal · 2019Article
- A PAGE screening approach for identifying CRISPR-Cas9-induced mutations in zebrafish.BioTechniques · 2018Article
- An optimised pipeline for parallel image-based quantification of gene expression and genotyping afterBiology open · 2018Article
- Genetic epidemiology of titin-truncating variants in the etiology of dilated cardiomyopathy.Biophysical reviews · 2017Review
- A rapid and effective method for screening, sequencing and reporter verification of engineered frameshift mutations in zebrafish.Disease models & mechanisms · 2017Article
- The Impact of CRISPR/Cas9 Technology on Cardiac Research: From Disease Modelling to Therapeutic Approaches.Stem cells international · 2017Review
- A simplified method for identifying early CRISPR-induced indels in zebrafish embryos using High Resolution Melting analysis.BMC genomics · 2016Article
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Authors and funding
7 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
The introduction of new genome editing tools such as ZFNs, TALENs and, more recently, the CRISPR/Cas9 system, has greatly expanded the ability to knock-out genes in different animal models, including zebrafish. However, time and costs required for the screening of a huge number of animals, aimed to identify first founder fishes (F0), and then carriers (F1) are still a bottleneck. Currently, high-resolution melting (HRM) analysis is the most efficient technology for large-scale InDels detection, but the very expensive equipment demanded for its application may represent a limitation for research laboratories. Here, we propose a rapid and cheap method for high-throughput genotyping that displays efficiency rate similar to the HRM. In fact, using a common ViiA™7 real-time PCR system and optimizing the parameters of the melting analysis, we demonstrated that it is possible to discriminate between the mutant and the wild type melting curves. Due to its simplicity, rapidity and cheapness, our method can be used as a preliminary one-step approach for massive screening, in order to restrict the scope at a limited number of embryos and to focus merely on them for the next sequencing step, necessary for the exact sequence identification of the induced mutation. Moreover, thanks to its versatility, this simple approach can be readily adapted to the detection of any kind of genome editing approach directed to genes or regulatory regions and can be applied to many other animal models.
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