- Zinc-finger nucleases (ZFNs) are engineered proteins that combine DNA-binding domains with a DNA-cleaving enzyme. The DNA-binding portion consists of zinc finger proteins, which are sequences of protein domains that each recognize specific DNA sequences. These zinc fingers are typically engineered to recognize sequences of 3 base pairs each, and multiple zinc fingers are combined to achieve greater specificity.
- The nuclease component typically comes from the FokI restriction enzyme, which functions as a dimer to create double-strand breaks in DNA. Each ZFN monomer contains 3-6 zinc finger modules linked to a FokI nuclease domain. Two ZFN monomers must bind their respective target sequences on opposite DNA strands for the FokI domains to dimerize and create a double-strand break.
- When the DNA break occurs, cellular repair mechanisms activate. The primary repair pathways are non-homologous end joining (NHEJ), which often results in small insertions or deletions, or homology-directed repair (HDR) if a donor DNA template is present. These repair outcomes can be exploited for genome editing purposes like gene knockout, gene correction, or gene insertion.
- ZFNs were among the first programmable nucleases used for genome editing, paving the way for later technologies like TALENs and CRISPR. Their key advantages include high specificity when well-designed and the ability to target nearly any DNA sequence. However, designing effective ZFNs requires significant expertise, their assembly can be technically challenging, and they may have more off-target effects compared to newer genome editing tools.
- Applications of ZFNs include creating knockout cell lines and animal models, engineering disease-resistant crops, and developing potential gene therapies for genetic disorders. Several clinical trials have explored ZFN-based treatments for diseases like HIV, hemophilia, and certain cancers.
