Zinc finger nucleases (ZFNs) are a class of engineered DNA-binding proteins that facilitate targeted editing of the genome by creating double-strand breaks in DNA at user-specified locations.
Figure 1.Each Zinc Finger Nuclease (ZFN) consists of two functional domains: a.) A DNA-binding domain comprised of a chain of two-finger modules, each recognizing a unique hexamer (6 bp) sequence of DNA. Two-finger modules are stitched together to form a Zinc Finger Protein, each with specificity of ≥ 24 bp. b.) A DNA-cleaving domain comprised of the nuclease domain of Fok I. When the DNA-binding and DNA-cleaving domains are fused together, a highly-specific pair of 'genomic scissors' are created.
Double-strand breaks are important for site-specific mutagenesis in that they stimulate the cell's natural DNA-repair processes, namely homologous recombination and non-homologous end joining (NHEJ). By implementing established, field proven methods, these processes are harnessed to generate precisely targeted genomic edits, resulting in cell lines with targeted gene deletions, integrations, or modifications.
Figure 2.ZFN-mediated genome editing takes place in the nucleus when a ZFN pair targeting the user’s gene of interest is delivered into a parental cell line, either by transfection, electroporation or viral delivery.