ZFN

Zinc finger nucleases (ZFN) are engineered restriction enzymes used for targeted genome editing. They are chimeric proteins that fuse a DNA-binding domain made of zinc finger motifs to the FokI nuclease domain. The DNA-binding domain can be designed to recognize a specific 9- to 18-base pair sequence, depending on the number of fingers used.

Mechanism: Two ZFN monomers bind to opposite DNA strands with a spacer; FokI nuclease domains dimerize and introduce a double-strand break (DSB) in the spacer. The DSB triggers cellular repair via non-homologous end joining or homology-directed repair.

Design and limitations: Zinc finger modules often show context-dependent binding; modular assembly can be straightforward but may require empirical screening. Off-target activity is a concern; multiple strategies exist to improve specificity. ZFNs can be delivered as DNA, RNA, or as protein-RNPs.

History and status: ZFNs were among the first programmable nucleases used for genome editing; prominent in the 2000s; prior to the CRISPR-Cas9 wave; though widely used in research and some therapeutics, they have been largely supplanted by TALENs and CRISPR-Cas systems, which are easier to design and retarget.

Applications: gene disruption, targeted gene insertion or replacement using donor templates; generation of knockout cell lines in model organisms; potential therapeutic strategies under development, subject to safety considerations.