Zinc-finger nuclease

Zinc-finger nuclease (ZFN) is a type of artificial restriction enzyme generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc-finger nucleases are important tools in genetic engineering for making precise alterations to an organism's DNA. This technology has been pivotal in the development of gene therapy and genetically modified organisms.

Overview

Zinc-finger nucleases (ZFNs) are synthetic restriction enzymes created by engineering a protein that can bind to specific DNA sequences and introduce double-strand breaks at specific locations within the genome. The specificity of ZFNs comes from the zinc finger domains, which can be designed to target virtually any desired DNA sequence. This specificity allows for targeted gene disruption, correction, or insertion.

Mechanism

The mechanism of action of ZFNs involves the recognition and binding of the zinc finger domain to a specific DNA sequence. Each zinc finger unit recognizes a 3-4 base pair sequence of DNA, and multiple zinc fingers can be assembled together to recognize longer sequences. Once bound to its target DNA, the FokI nuclease domain—fused to the zinc finger domain—dimerizes and introduces a double-strand break in the DNA. This break can then be repaired by the cell's natural DNA repair mechanisms, either non-homologous end joining (NHEJ) or homology-directed repair (HDR), leading to gene disruption or precise gene editing, respectively.

Applications

ZFNs have been used in a wide range of applications, including:

• Gene therapy: ZFNs have been explored as tools for correcting genetic defects in human cells. For example, they have been used in attempts to correct the mutation responsible for sickle cell disease.
• Creation of genetically modified organisms: ZFNs have been used to introduce desirable traits into plants and animals, such as disease resistance or enhanced nutritional content.
• Functional genomics: ZFNs allow researchers to study the function of genes by creating targeted gene knockouts.

Advantages and Limitations

The primary advantage of ZFNs is their ability to make precise, targeted changes to the genome, which is not easily achievable with traditional genetic engineering techniques. However, the design and assembly of custom ZFNs can be technically challenging and expensive. Additionally, off-target effects, where the ZFNs bind and cleave unintended parts of the genome, can lead to unwanted mutations.

See Also

• CRISPR
• TALENs
• Genome editing