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Updated: May 30, 2026
09:11
Genome Editing with CompoZr Custom Zinc Finger Nucleases (ZFNs)
Published on: June 14, 2012
Zinc-finger recombinase activities in vitro
Marko M Prorocic1, Dong Wenlong, Femi J Olorunniji
1Institute of Infection, Immunity and Inflammation, University of Glasgow, GBRC, Glasgow G12 8QQ, Scotland, UK.
Nucleic Acids Research
|August 19, 2011
Summary
Zinc-finger recombinases (ZFRs) efficiently catalyze site-specific DNA recombination and cleavage at engineered Z-sites. ZFR activity and specificity are influenced by Z-site core length and ZFR protein design, offering potential for genetic applications.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetic Engineering
Background:
- Zinc-finger recombinases (ZFRs) are engineered proteins combining DNA-binding zinc-finger domains with serine recombinase catalytic domains.
- ZFRs are designed for site-specific DNA recombination at user-defined Z-sites.
Purpose of the Study:
- To characterize the in vitro and in vivo recombination activity and specificity of ZFRs.
- To investigate the influence of Z-site core sequence length and ZFR protein design on recombination efficiency.
Main Methods:
- Purification and in vitro characterization of ZFRs.
- Assays for site-specific DNA recombination and cleavage in vitro and in Escherichia coli.
- Analysis of ZFR activity under varying Z-site lengths and ZFR linker lengths.
Main Results:
- Purified ZFRs demonstrated highly specific and efficient reciprocal recombination between Z-sites in vitro, with no detectable off-site activity.
- ZFR activity was highly dependent on Z-site core sequence length, affecting binding, synapsis, and DNA cleavage.
- ZFRs can also mediate Z-site-specific double-strand DNA cleavage.
- A short peptide linker (2 amino acids) in ZFR design enhanced in vitro activity and sensitivity to Z-site length, unlike a long linker.
Conclusions:
- ZFRs are potent tools for precise DNA manipulation, exhibiting high specificity and efficiency.
- The length of the Z-site core sequence and the ZFR protein's linker region are critical determinants of recombination activity and specificity.
- These findings have significant implications for the development and application of ZFR technology in genetic engineering and synthetic biology.
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