Harel, Itamar ORCID: 0000-0001-9749-8279, Valenzano, Dario Riccardo ORCID: 0000-0002-8761-8289 and Brunet, Anne (2016). Efficient genome engineering approaches for the short-lived African turquoise killifish. Nat. Protoc., 11 (10). S. 2010 - 2029. LONDON: NATURE PUBLISHING GROUP. ISSN 1750-2799

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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.

Item Type: Journal Article
CreatorsEmailORCIDORCID Put Code
Harel, ItamarUNSPECIFIEDorcid.org/0000-0001-9749-8279UNSPECIFIED
Valenzano, Dario RiccardoUNSPECIFIEDorcid.org/0000-0002-8761-8289UNSPECIFIED
URN: urn:nbn:de:hbz:38-261727
DOI: 10.1038/nprot.2016.103
Journal or Publication Title: Nat. Protoc.
Volume: 11
Number: 10
Page Range: S. 2010 - 2029
Date: 2016
Place of Publication: LONDON
ISSN: 1750-2799
Language: English
Faculty: Unspecified
Divisions: Unspecified
Subjects: no entry
Uncontrolled Keywords:
Biochemical Research MethodsMultiple languages
Refereed: Yes
URI: http://kups.ub.uni-koeln.de/id/eprint/26172


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