Zinc finger-based knockout punches for zebrafish genes

Abstract

The ability to manipulate the genome is critical to develop and test hypotheses based on genetics. Knockdown strategies focused on RNAi and/or morpholinos are excellent genetic tools, but they come with substantial technical limitations. A new gene targeting approach employing synthetic zinc finger nuclease (ZFN) technology is a powerful and complementary approach to directly modify genetic loci for many diverse applications, notably enhancing Danio rerio (the zebrafish) as an experimental organism for understanding human disease. This ZFN-based technology to generate targeted knockouts in this aquatic animal opens the door to an array of new biological models of human disease and genetic testing.

FIG. 1.

Engineered ZFN cartoon. Total sequence specificity is derived from individual ZFN1 and ZFN2 fingers. The FokI endonuclease domain introduces double-stranded breaks after sequence-specific DNA binding.

FIG. 2.

How to use ZFNs to make a targeted gene knockout in zebrafish.^, mRNA encoding the selected ZFN is injected into one-cell zebrafish embryos. This custom ZFN binds and cuts at the specified locus and cell repairs this DNA break imprecisely to introduce mutations at the selected gene. The specific molecular lesion is clonally selected and determined after genotyping the offspring from these mosaic founders.