title: Efficient genome engineering approaches for the short-lived African turquoise killifish.
abstract: A central challenge in experimental aging research is the lack of short-lived vertebrate models for genetic studies. Here we present a comprehensive protocol for efficient genome engineering in the African turquoise killifish (Nothobranchius furzeri), which is the shortest-lived vertebrate in captivity with a median life span of 4-6 months. By taking advantage of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease (CRISPR/Cas9) system and the turquoise killifish genome, this platform enables the generation of knockout alleles via nonhomologous end joining (NHEJ) and knock-in alleles via homology-directed repair (HDR). We include guidelines for guide RNA (gRNA) target design, embryo injection and hatching, germ-line transmission and for minimizing off-target effects. We also provide strategies for Tol2-based transgenesis and large-scale husbandry conditions that are critical for success. Because of the fast life cycle of the turquoise killifish, stable lines can be generated as rapidly as 2-3 months, which is much faster than other fish models. This protocol provides powerful genetic tools for studying vertebrate aging and aging-related diseases.
authors.affiliation: Department of Genetics, Stanford University, Stanford, California, USA.
authors.forename: Itamar
authors.initials: I
authors.lastname: Harel
authors.affiliation: Max Planck Institute for Biology of Ageing, Cologne, Germany.|CECAD, University of Cologne, Cologne, Germany.
authors.forename: Dario Riccardo
authors.initials: DR
authors.lastname: Valenzano
authors.affiliation: Department of Genetics, Stanford University, Stanford, California, USA.|Glenn Laboratories for the Biology of Aging at Stanford, Stanford, California, USA.
authors.forename: Anne
authors.initials: A
authors.lastname: Brunet