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

Developers

Itamar Harel, Dario Riccardo Valenzano, Anne Brunet.

Description of the technology

A central challenge in experimental aging research is the lack of short-lived vertebrate models for genetic studies.

This technology proposes 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, platform of this technology enables the generation of knockout alleles via nonhomologous end joining (NHEJ) and knock-in alleles via homology-directed repair (HDR). The technology includes guidelines for guide RNA (gRNA) target design, embryo injection and hatching, germ-line transmission and for minimizing off-target effects. The technology also provides strategies for Tol2-based transgenesis and large-scale husbandry conditions that are critical for success.

Practical application

The technology is highly promising for use in the investigations of ageing. It allows to study sophisticated processes of gene changes in process of ageing.

Because of the fast life cycle of the turquoise killifish, stable lines can be generated, using this technology, as rapidly as 2–3 months, which is much faster than other fish models. This protocol provides powerful genetic tools (e.g., CRISPR/Cas9 system) in combination with short-lived model for studying vertebrate aging and aging-related diseases.

Laboratories

• Department of Genetics, Stanford University, Stanford (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 (USA)

Links

Publications

• Harel, I., Valenzano, D.R., Brunet, A. «Efficient genome engineering approaches for the short-lived African turquoise killifish." 11 Nature Protocols (2016): 2010–2028.
• Harel, I. & Brunet, A. «The African turquoise killifish: a model for exploring vertebrate aging and diseases in the fast lane." 80 Cold Spring Harb. Symp. Quant. Biol. (2015): 275–279.
• Harel, I. et al. «A platform for rapid exploration of aging and diseases in a naturally short-lived vertebrate." 160 Cell, (2015): 1013–1026.
• Valenzano, D.R. et al. «The African turquoise killifish genome provides insights into evolution and genetic architecture of lifespan." 163 Cell, (2015): 1539–1554.