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

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

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


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.


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