Universität zu Köln

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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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.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Harel, Itamar UNSPECIFIED orcid.org/0000-0001-9749-8279 UNSPECIFIED Valenzano, Dario Riccardo UNSPECIFIED orcid.org/0000-0002-8761-8289 UNSPECIFIED Brunet, Anne UNSPECIFIED UNSPECIFIED UNSPECIFIED
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
Publisher:	NATURE PUBLISHING GROUP
Place of Publication:	LONDON
ISSN:	1750-2799
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language Biochemical Research Methods Multiple languages
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/26172