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

Nature Biotechnology volume 25, pages 786–793 (2007)Cite this article

Abstract

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 describe a structure-based approach to reducing off-target cleavage. Using in silico protein modeling and energy calculations, we increased the specificity of target site cleavage by preventing homodimerization and lowering the dimerization energy. Cell-based recombination assays confirmed that the modified ZFNs were as active as the original ZFNs but elicit significantly less genotoxicity. The improved safety profile may facilitate therapeutic application of the ZFN technology.

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Figure 1: Structure-based redesign of the FokI dimer interface.
Figure 2: In vitro cleavage specificity.
Figure 3: Single strand annealing assay.
Figure 4: Stimulation of homologous recombination with episomal and chromosomal target locus.
Figure 5: Stimulation of chromosomal homologous recombination by single and double variant ZFNs.
Figure 6: ZFN-associated genotoxicity.

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Acknowledgements

We thank Eva Guhl for technical assistance, Ian Korf for his assistance with genomic searches, Tatjana Cornu for critical reading of the manuscript, and Caroline Lilley, Matthew Weitzman and Regine Heilbronn for valuable discussions. This work was supported by grants CA311/1-2 and CA311/2-1 from the German Research Foundation, DFG (T.C.), grant CA103651 from the National Cancer Institute, National Institutes of Health (D.J.S.), an EC NEST grant, Netsensor (L.S.), and an equipment grant from the Sonnenfeld-Stiftung, Berlin (T.C.).

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Author notes

  1. Michal Szczepek, Vincent Brondani and Janine Büchel: These authors contributed equally to this work.

Authors and Affiliations

  1. Charité Medical School, Institute of Virology (CBF), Berlin, 12203, Germany

    Michal Szczepek, Janine Büchel & Toni Cathomen

  2. Genome Center and Department of Pharmacology, University of California Davis, Davis, 95616, California, USA

    Vincent Brondani & David J Segal

  3. Centre de Regulacio Genomica, CRG-EMBL Systems Biology Unit, Barcelona, 08003, Spain

    Luis Serrano

Authors
  1. Michal Szczepek
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  2. Vincent Brondani
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Contributions

L.S. performed protein modeling and in silico mutagenesis; M.S. and V.B. generated the single variant ZFNs; V.B. performed in vitro cleavage and SSA assays; M.S. carried out HR-based assays; J.B. generated the double variant ZFNs, performed immunoblotting and immunofluorescence, and established the genotoxicity assay; D.J.S. and T.C. conceived and directed the project, supervised M.S., V.B. and J.B., and wrote the manuscript.

Corresponding authors

Correspondence to David J Segal or Toni Cathomen.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Amino acid sequence of GZF1-N and GZF3-N. (DOC 25 kb)

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Szczepek, M., Brondani, V., Büchel, J. et al. Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nat Biotechnol 25, 786–793 (2007). https://doi.org/10.1038/nbt1317

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