Designer molecular scissors for genome surgery. A schematic representation of a pair of 4-finger ZFNs (zinc finger nucleases) bound to their cognate sites in the IL2Rγ gene [4]. Since a pair of ZFNs requires two copies of the 12-bp recognition sites in a tail-to-tail orientation, they effectively have a 24-bp recognition site, which is long enough to specify a unique genomic address in human cells. Since the ZFN-binding sites in the IL2Rγ gene are not identical, both ZFNs that bind these sites need to be introduced into the cell to induce a targeted genomic DSB. The ZFN sites in the IL2Rγ gene are separated by 5 bp [4].

Schematic representation of two of several possible mechanisms for a DSB repair (HR and NHEJ) in mammalian cells. The DSB (double-strand break) may arise spontaneously such as by damage from reactive oxygen species during normal metabolism, induced randomly by the exposure to ionizing radiation, or induced specifically by zinc finger nucleases. The repair of the gene by homologous recombination (HR) occurs after the induction of a DSB. During HR, the DSB is processed to form free 3′ single-strand tails, a process that requires the Mre11/Rad50/Nbs1 complex [61]. The HR machinery, through the actions of the strand invasion protein Rad51, then uses the free 3′ ends to invade a homologous repair template/donor. How the machinery identifies a homologous repair donor remains unclear, but it is likely that simple physical proximity plays an important role. In the normal repair of a DSB, the repair donor is the sister chromatid, and thus the template is identical to the damaged allele. In gene targeting, the repair donor would be an extrachromosomal piece of DNA that could have sequence differences. After strand invasion, primed DNA synthesis occurs to generate new undamaged DNA using the undamaged donor DNA template. The process is completed by the annealing of the new strand of DNA with its original partner and subsequent use of that new DNA to template DNA synthesis. During non-homologous end joining (NHEJ), the broken ends from the DSB are bound by specific protein factors to limit nucleolytic degradation. The ends are then bridged together by protein-protein interactions between the protein factors. The sequences are then aligned and ligated [62]. NHEJ often occurs without any homology or even microhomologies between the ends that are ligated. NHEJ is mutagenic by nature because it often introduces small deletions or insertions at the fusion site.

A schematic representation of ZFN-mediated gene targeting in human cells. Targeted ZFN-mediated disruption of the Gene X by NHEJ occurs when cells are transfected with designer ZFNs alone. The ZFNs induce a targeted genomic DSB in cells, which stimulates NHEJ that is mutagenic by nature. It gives rise to a pool of different Gene X mutants (depicted as mGene X). Targeted disruption of the Gene X by ZFN-evoked HR occurs when the normal cells are transfected with both designer ZFNs and the homologous donor DNA containing a predetermined Gene X modification to generate a specific desired mutant (depicted as smGene X) of the targeted gene. The ZFNs induce a targeted genomic DSB in cells, which stimulates local HR in the presence of homologous donor DNA, the recombinogenic repair pathway. Alternatively, to achieve targeted gene correction (or gene editing), the mutant Gene X cells are transfected with both designer ZFNs and the homologous correcting donor DNA fragment of the target gene to generate normal cells.