期刊
NATURE PROTOCOLS
卷 11, 期 10, 页码 2010-2028出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2016.103
关键词
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资金
- US National Institutes of Health (NIH) [DP1AG044848]
- Glenn Laboratories for the Biology of Aging
- Max Planck Society
- Max Planck Institute for Biology of Ageing
- Damon Runyon fellowship
- Rothschild fellowship
- HFSP fellowship
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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