Efficient genome engineering approaches for the short-lived African turquoise killifish

Itamar Harel; Dario Riccardo Valenzano; Anne Brunet

doi:10.1038/nprot.2016.103

Efficient genome engineering approaches for the short-lived African turquoise killifish

Nat Protoc. 2016 Oct;11(10):2010-2028. doi: 10.1038/nprot.2016.103. Epub 2016 Sep 22.

Authors

Itamar Harel¹, Dario Riccardo Valenzano^{2

3}, Anne Brunet^{1

4}

Affiliations

¹ Department of Genetics, Stanford University, Stanford, California, USA.
² Max Planck Institute for Biology of Ageing, Cologne, Germany.
³ CECAD, University of Cologne, Cologne, Germany.
⁴ Glenn Laboratories for the Biology of Aging at Stanford, Stanford, California, USA.

PMID: 27658015
DOI: 10.1038/nprot.2016.103

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.