Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases

@article{Szczepek2007StructurebasedRO,
  title={Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases},
  author={Michal Szczepek and Vincent Brondani and Janine B{\"u}chel and Luis Serrano and David J. Segal and Toni Cathomen},
  journal={Nature Biotechnology},
  year={2007},
  volume={25},
  pages={786-793}
}
Artificial endonucleases consisting of a FokI cleavage domain tethered to engineered zinc-finger DNA-binding proteins have proven useful for stimulating homologous recombination in a variety of cell types. Because the catalytic domain of zinc-finger nucleases (ZFNs) must dimerize to become active, two subunits are typically assembled as heterodimers at the cleavage site. The use of ZFNs is often associated with significant cytotoxicity, presumably due to cleavage at off-target sites. Here we… 

Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures

The development and application of a yeast-based selection system designed to functionally interrogate the ZFN dimer interface is reported, identified critical residues involved in dimerization through the isolation of cold-sensitive nuclease domains, and used to engineer ZFNs that have superior cleavage activity while suppressing homodimerization.

Attenuation of Zinc Finger Nuclease Toxicity by Small-Molecule Regulation of Protein Levels

By regulating protein levels, it is shown that by maintaining high rates of ZFN-mediated gene targeting while reducing ZFN toxicity, the cytotoxic effects currently associated with many ZFNs can be maintained.

Creating designed zinc-finger nucleases with minimal cytotoxicity.

DNA-binding specificity is a major determinant of the activity and toxicity of zinc-finger nucleases.

The results of these cell-based assays reveal that the DNA-binding specificity--in addition to the affinity--is a major determinant of ZFN activity and is inversely correlated with ZFN-associated toxicity, and provide the first evidence that engineering strategies, which account for context-dependent DNA- binding effects, yield ZFs that function as highly efficient ZFNs in human cells.

DNA-binding Specificity Is a Major Determinant of the Activity and Toxicity of Zinc-finger Nucleases.

The results of these cell-based assays reveal that the DNA-binding specificity-in addition to the affinity-is a major determinant of ZFN activity and is inversely correlated with ZFN-associated toxicity, and provide the first evidence that engineering strategies, which account for context-dependent DNA- binding effects, yield ZFs that function as highly efficient ZFNs in human cells.

Zinc-finger recombinase activities in vitro

It is shown that purified ZFRs catalyse efficient high-specificity reciprocal recombination between pairs of Z-sites in vitro, and that the design of the ZFR protein itself is also a crucial variable affecting activity.

Quantification of zinc finger nuclease-associated toxicity.

Two different methods to quantify ZFN-associated toxicity are described: the genotoxicity assay is based on quantification of DSB repair foci induced by ZFNs whereas the cytotoxicity is based upon assessing cell survival after application of ZFNS.

Autonomous zinc-finger nuclease pairs for targeted chromosomal deletion

It is demonstrated that two autonomous ZFN pairs can be directed simultaneously to two different sites to induce a chromosomal deletion in ∼10% of alleles and will prove useful in targeted genome engineering approaches wherever an application requires the expression of two distinct Z FN pairs.

In vitro assessment of zinc finger nuclease activity.

A simple and fast method for the functional testing of ZFNs in vitro, including the specificity of DNA binding, the kinetics of dimerization of the two ZFN subunits, and the catalytic activity is presented.
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