Gene Function Validation via Zinc Finger Nuclease
- Zinc-finger nucleases (ZFNs) are synthetic restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger domains can be engineered to target desired DNA sequences and this enables zinc-finger nucleases to target unique sequences within complex genomes. By taking advantage of endogenous DNA repair machinery, these reagents can be used to precisely alter the genomes of higher organisms.
- FokI is typically used as the cleavage domain in ZFNs. This cleavage domain must dimerize in order to cleave DNA and thus a pair of ZFNs are required to target non-palindromic DNA sites. Standard ZFNs fuse the cleavage domain to the C-terminus of each zinc finger domain. In order to allow the two cleavage domains to dimerize and cleave DNA, the two individual ZFNs must bind opposite strands of DNA with their C-termini a defined distance apart. The most commonly used linker sequences between the zinc finger domain and the cleavage domain requires the 5′ edge of each binding site to be separated by 5 to 7 bp. The non-specific cleavage domain from the Type IIS restriction endonuclease
- The Cys2His2 zinc finger is a module of about 28-30 amino acids that is found in a large family of eukaryotic transcription factors. Its structure consists of an a-helix, two b-sheets, and a single zinc atom. Each finger recognizes principally three base pairs of DNA through contacts in the major groove.
- The cleavage domain from FokI has to dimerize to be active as a nuclease. The dimer interface is quite weak – the protein is essentially never seen as a dimer in solution. This turns out to be very beneficial, as a monomeric ZFN is not active; the cleavage reagent is assembled at the dual target site by binding of separate molecules, each of which must recognize its binding site.
- ZFNs have been shown to be functional and efficient at cleaving synthetic and genomic targets in Drosophila, mammalian cells, C. elegans, Arabidopsis, cultured plant cells, and Xenopus oocytes. Because double-strand breaks in DNA stimulate both inaccurate repair and homologous recombination in essentially all organisms, the ZFN procedures should be very broadly applicable. ZFNs technology was named as the “Method of the year in 2010” by Nature Methods.
Designing ZFNs for Novel Gene Targets
The basic steps involved in designing ZFN for gene knock out are as follows:
- Choose a target gene or other DNA sequence of interest.
- Search this sequence for sites comprised of triplets for which good zinc fingers already exist.
- Clone each ZFP coding sequence in frame with the FokI cleavage domain.
- Transfer the resulting ZFN coding sequence to an expression vector appropriate for the intended use.
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- Bibikova, M., Beumer, K., Trautman, J.K. and Carroll, D. (2003). Enhancing gene targeting with designed zinc-finger nucleases. Science 300: 764.
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