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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.
The zebrafish (Danio rerio) is the premier nonmammalian vertebrate model organism with a nearly complete genome sequenced by the Sanger Center. This small aquatic fish is being utilized by over 1000 laboratories around the world because of its biological similarity to humans, its advanced molecular genetics, the ready access to its genome sequence, and the ever-expanding and outstanding new biological tools now available to the zebrafish researcher. The fish is an ideal system for studying processes difficult or impossible to follow in other animals such as the mechanisms underlying organogenesis. Unlike mice, this model system is readily amenable to forward genetic mutagenesis approaches for the identification of new genes required for these key developmental processes and functions. In the areas of vertebrate development biology and functional genomics, the transparency of the zebrafish embryo during development has allowed researchers to track regulation of gene expression using fluorescent proteins in real time in living animals. Consequently, the conversion of genetic and other biological information learned from the fish to humans has been faster than in other vertebrate systems. The major missing component has been an effective reverse genetic tool for targeted knockouts. Zinc finger nucleases (ZFNs) are a powerful new molecular method for making these gene knockouts in the zebrafish and likely other model organisms.
ZFNs are engineered restriction enzymes that can be customized to cut the DNA sequence of interest (Fig. 1; see Porteus and Carroll1 for review). This is a platform technology with many potential uses from somatic cell work for modifying tissue culture cells in vitro to gene therapy applications (see Porteus and Carroll1 for review).
Perhaps the most powerful market for this tool is its use in germline gene modification. Recent experiments show that the application of ZFNs against zebrafish genes results in an impressive>25% of offspring with germline modification of the exon targeted by the customized gene-specific ZFN.2,3
Zebrafish embryos are injected with the custom ZFN-encoding mRNA, reared, and out-crossed (Fig. 2). A simple fin clip, PCR, and sequencing genotyping process determines the precise nature of the induced mutation in the altered chromosome, typically resulting in a frameshift allele.2,3
These two papers used different ZFN platforms for the generation of custom ZFNs.2,3 Doyon et al. used the proprietary technology from their collaborators at Sangamo, while Meng et al. deployed an academic-based library. Neither library of vetted ZFNs is currently very complex, making the description of methods for the generation of new custom ZFNs a critical aspect of these papers. Access to both tested ZFNs and the process to make more is described in both papers.
An ideal strategy would be to develop a sufficiently complex, off-the-shelf collection of tested ZFNs for deployment on a genome-wide scale. Such fingers could be then placed virtually on the zebrafish genome database with a focus on unique sites within exons for those interested in the generation of new knockouts. Optimism for such an approach comes from efforts of the Zinc Finger Consortium, a group of academic scientists working to develop facile and robust open-access platforms for engineering customized zinc finger arrays through modular design and selection.4
The ZFN approach looks very good, and has clear advantages of reduced collateral genetic damage found in TILLING methods. However, ZFN technology has yet to be scaled up. We also do not know yet whether all genes will be accessible via this method. Regardless of the approach, all of these technologies share in the common goal of using genetic methods to address important problems in biology. The more tools we have, the better for testing complex scientific hypotheses. How will ZFNs fit into the overall mix? Stay tuned.
Many thanks to Dr. Dan Voytas for Figure 1 and for many stimulating discussions on this topic. This manuscript was supported in part by NIH grants to S.C.E. (GM63904, DA14546).