Generating highly specific nucleases by a fusion approach

In the post-genomic era, highly specific nucleases became important tools for directed gene targeting and gene therapy. They should be used to specifically cleave a predefined sequence in a gene affected in a monogenetic disease. By creating a double strand break, homologous recombination, an otherwise rare event in the cell, will be stimulated manifold. Most commonly used are zinc-finger nucleases, consisting of specific zinc-finger modules, responsible for recognizing a specific DNA sequence, fused to the catalytic domain of the restriction enzyme FokI. Recently, so-called TALE-nucleases have been produced for the same purpose. In the study presented here, a highly specific nuclease was created by using a catalytically inactive variant of a homing endonuclease (HE) as a DNA-binding module fused to a type IIP restriction enzyme (RE) as a cleavage module. The use of a specific RE instead of only the catalytic domain of FokI as cleavage module has the advantage that the RE also contributes to the specificity of the fusion enzyme, while the catalytic domain of FokI by itself does not specifically bind to DNA. By optimizing the linker between binding- and cleavage module and introducing several mutations in both enzymes a variant was obtained, cleaving addressed sites (RE site flanked by two HE sites) in vitro with an over 1000-fold preference over unaddressed sites (RE site only). This enzyme may present an alternative to the above described gene targeting nucleases.