Developing a novel zinc-finger nuclease with a sequence-specific cleavage module

Conventional zinc-finger nucleases (ZFNs) typically consist of three to four zinc-fingers (ZFs) and the non-specific DNA cleavage domain of the restriction endonuclease FokI. In gene targeting approaches, ZFNs have been successfully used for genetic modifications within different organisms and cells via double strand break induced homology-directed repair. However, it has been reported that ZFNs also show unwanted cleavage at so called "off-target" sites in vivo, leading to ZFN-mediated toxicity. In order to add an extra element of specificity, we have fused three ZFs to the restriction endonuclease PvuII to generate an alternative ZFN with a sequence-specific cleavage module. ZF-PvuII constructs must be designed such that only PvuII sites with adjacent ZF binding sites are cleaved. To achieve this, we selected suitable linkers and introduced amino acid substitutions into PvuII that alter K_m and k_cat and increase fidelity. The resulting ZF-PvuII constructs were tested for their activity and specificity in vitro. The optimized ZF-PvuII fusion constructs cleave DNA at addressed sites with a more than 1000-fold preference over unaddressed PvuII sites. Even at excess of enzyme over substrate and prolonged incubation times, no unaddressed cleavage of ZF-PvuII was detected, in contrast to an observed unspecific cleavage of the corresponding ZFN with the FokI cleavage module. These results present the ZF-PvuII platform as a valid alternative to conventional ZFNs.