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

Area of Science:

• Genetics
• Aging Research
• Comparative Genomics

Background:

• Experimental aging research requires short-lived vertebrate models for genetic studies.
• The African turquoise killifish (Nothobranchius furzeri) is the shortest-lived vertebrate, with a median lifespan of 4-6 months.
• Existing genetic tools for short-lived vertebrates are limited.

Purpose of the Study:

• To present a comprehensive protocol for efficient genome engineering in Nothobranchius furzeri.
• To establish a platform for generating knockout and knock-in alleles using CRISPR/Cas9.
• To facilitate the study of vertebrate aging and aging-related diseases.

Main Methods:

• Utilized the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9 nuclease (CRISPR/Cas9) system.
• Employed nonhomologous end joining (NHEJ) for knockout allele generation.
• Used homology-directed repair (HDR) for knock-in allele generation.
• Incorporated Tol2-based transgenesis and optimized large-scale husbandry.
• Provided guidelines for guide RNA (gRNA) design, embryo manipulation, and minimizing off-target effects.

Main Results:

• Efficient genome engineering in Nothobranchius furzeri was achieved.
• Stable transgenic lines were generated in 2-3 months, significantly faster than other fish models.
• The protocol enables both knockout and knock-in allele generation.

Conclusions:

• This protocol provides powerful genetic tools for aging research using the African turquoise killifish.
• The rapid generation of genetically modified killifish accelerates studies on aging and age-related diseases.
• Nothobranchius furzeri serves as an effective model for rapid genetic manipulation in vertebrates.