title: A Rapid and Cheap Methodology for CRISPR/Cas9 Zebrafish Mutant Screening.
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.
authors.affiliation: Biology and Evolution of Marine Organisms (BEOM), Stazione Zoologica Anton Dohrn, Villa Comunale, 80121, Naples, Italy.
authors.forename: Ylenia
authors.initials: Y
authors.lastname: D'Agostino
authors.affiliation: Biology and Evolution of Marine Organisms (BEOM), Stazione Zoologica Anton Dohrn, Villa Comunale, 80121, Naples, Italy.
authors.forename: Annamaria
authors.initials: A
authors.lastname: Locascio
authors.affiliation: Biology and Evolution of Marine Organisms (BEOM), Stazione Zoologica Anton Dohrn, Villa Comunale, 80121, Naples, Italy.
authors.forename: Filomena
authors.initials: F
authors.lastname: Ristoratore
authors.affiliation: Biology and Evolution of Marine Organisms (BEOM), Stazione Zoologica Anton Dohrn, Villa Comunale, 80121, Naples, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Sordino
authors.affiliation: Biology and Evolution of Marine Organisms (BEOM), Stazione Zoologica Anton Dohrn, Villa Comunale, 80121, Naples, Italy.
authors.forename: Antonietta
authors.initials: A
authors.lastname: Spagnuolo
authors.affiliation: Research Infrastructures for Marine Biological Resources (RIMAR), Stazione Zoologica Anton Dohrn, Villa Comunale, 80121, Naples, Italy. borra@szn.it.
authors.forename: Marco
authors.initials: M
authors.lastname: Borra
authors.affiliation: Biology and Evolution of Marine Organisms (BEOM), Stazione Zoologica Anton Dohrn, Villa Comunale, 80121, Naples, Italy. salvatore.daniello@szn.it.
authors.forename: Salvatore
authors.initials: S
authors.lastname: D'Aniello
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics*; D055879:Gene Knock-In Techniques / Q000379:methods*; D055786:Gene Knockout Techniques / Q000379:methods*; D016678:Genome; D005838:Genotype; D009154:Mutation / Q000235:genetics*; D015027:Zebrafish / Q000235:genetics
keywords: CRISPR/Cas9; Derivative melting curve; Mutation screening; Zebrafish knock-out mutants; qPCR