Zinc Finger Nucleases: A new era for transgenic animals

The rational engineering of eukaryotic genomes would facilitate the study of heritable changes in gene expression and offer enormous potential across basic research, drug-discovery, bioproduction and therapeutic development. A significant advancement toward this objective was achieved with the advent of a novel technology that enables high-frequency and high-fidelity genome editing via the application of custom designed zinc finger nucleases (ZFNs). A ZFN is a chimeric protein that consists of the non-specific endonuclease domain of FokI fused to a DNA-binding domain composed of an engineered zinc-finger motif. Within these chimeric proteins, the DNA binding specificity of the zinc finger protein determines the site of nuclease action. Once the engineered ZFNs recognize and bind to their specified locus, it leads to the dimerization of the two nuclease domains on the ZFNs to evoke a double-strand break (DSB) in the targeted DNA. The cell then employs the natural DNA repair processes of either non-homologous end joining (NHEJ) or homology-directed repair (HDR) to repair the targeted break. Due to the imperfect fidelity of NHEJ, a proportion of DSBs within a ZFN-treated cellular population will be misrepaired, leading to cells in which variable heterogeneous genetic insertions or deletions have been made at the target site. Alternatively, the HDR repair pathway enables precise insertion of a transgene or other defined alterations into the targeted region. By this approach, a donor template containing the transgene flanked by sequences that are homologous to the regions either side of the cleavage site is co-delivered into the cell along with the ZFNs. By creating a specific DSB, these cellular repair mechanisms are harnessed to generate precisely targeted genomic edits resulting in both cell lines and animal models with targeted gene deletions, integrations, or modifications. This review will discuss the development, mechanism of action, and applications of ZFN technology to genome engineering and the creation of animal models.