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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References
Vasquez, K.M., Marburger, K., Intody, Z. & Wilson, J.H. Manipulating the mammalian genome by homologous recombination. Proc. Natl. Acad. Sci. USA 98, 8403–8410 (2001).
Urnov, F.D. et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435, 646–651 (2005).
Rouet, P., Smih, F. & Jasin, M. Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease. Mol. Cell. Biol. 14, 8096–8106 (1994).
Choulika, A., Perrin, A., Dujon, B. & Nicolas, J.F. Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of Saccharomyces cerevisiae. Mol. Cell. Biol. 15, 1968–1973 (1995).
Epinat, J.C. et al. A novel engineered meganuclease induces homologous recombination in yeast and mammalian cells. Nucleic Acids Res. 31, 2952–2962 (2003).
Porteus, M.H. Mammalian gene targeting with designed zinc finger nucleases. Mol. Ther. 13, 438–446 (2006).
Porteus, M.H. & Baltimore, D. Chimeric nucleases stimulate gene targeting in human cells. Science 300, 763 (2003).
Alwin, S. et al. Custom zinc-finger nucleases for use in human cells. Mol. Ther. 12, 610–617 (2005).
Kim, Y.G., Cha, J. & Chandrasegaran, S. Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc. Natl. Acad. Sci. USA 93, 1156–1160 (1996).
Liu, Q., Xia, Z., Zhong, X. & Case, C.C. Validated zinc finger protein designs for all 16 GNN DNA triplet targets. J. Biol. Chem. 277, 3850–3856 (2002).
Segal, D.J., Dreier, B., Beerli, R.R. & Barbas, C.F., III . Toward controlling gene expression at will: selection and design of zinc finger domains recognizing each of the 5′-GNN-3′ DNA target sequences. Proc. Natl. Acad. Sci. USA 96, 2758–2763 (1999).
Dreier, B., Beerli, R.R., Segal, D.J., Flippin, J.D. & Barbas, C.F., III . Development of zinc finger domains for recognition of the 5′-ANN-3′ family of DNA sequences and their use in the construction of artificial transcription factors. J. Biol. Chem. 276, 29466–29478 (2001).
Dreier, B. et al. Development of zinc finger domains for recognition of the 5′-CNN-3′ family DNA sequences and their use in the construction of artificial transcription factors. J. Biol. Chem. 280, 35588–35597 (2005).
Blancafort, P., Magnenat, L. & Barbas, C.F., III . Scanning the human genome with combinatorial transcription factor libraries. Nat. Biotechnol. 21, 269–274 (2003).
Hurt, J.A., Thibodeau, S.A., Hirsh, A.S., Pabo, C.O. & Joung, J.K. Highly specific zinc finger proteins obtained by directed domain shuffling and cell-based selection. Proc. Natl. Acad. Sci. USA 100, 12271–12276 (2003).
Smith, J. et al. Requirements for double-strand cleavage by chimeric restriction enzymes with zinc finger DNA-recognition domains. Nucleic Acids Res. 28, 3361–3369 (2000).
Bitinaite, J., Wah, D.A., Aggarwal, A.K. & Schildkraut, I. FokI dimerization is required for DNA cleavage. Proc. Natl. Acad. Sci. USA 95, 10570–10575 (1998).
Bibikova, M. et al. Stimulation of homologous recombination through targeted cleavage by chimeric nucleases. Mol. Cell. Biol. 21, 289–297 (2001).
Silva, G.H., Belfort, M., Wende, W. & Pingoud, A. From monomeric to homodimeric endonucleases and back: engineering novel specificity of LAGLIDADG enzymes. J. Mol. Biol. 361, 744–754 (2006).
Sims, P.A., Menefee, A.L., Larsen, T.M., Mansoorabadi, S.O. & Reed, G.H. Structure and catalytic properties of an engineered heterodimer of enolase composed of one active and one inactive subunit. J. Mol. Biol. 355, 422–431 (2006).
Bolon, D.N., Grant, R.A., Baker, T.A. & Sauer, R.T. Specificity versus stability in computational protein design. Proc. Natl. Acad. Sci. USA 102, 12724–12729 (2005).
Wah, D.A., Hirsch, J.A., Dorner, L.F., Schildkraut, I. & Aggarwal, A.K. Structure of the multimodular endonuclease FokI bound to DNA. Nature 388, 97–100 (1997).
Wah, D.A., Bitinaite, J., Schildkraut, I. & Aggarwal, A.K. Structure of FokI has implications for DNA cleavage. Proc. Natl. Acad. Sci. USA 95, 10564–10569 (1998).
Schymkowitz, J.W. et al. Prediction of water and metal binding sites and their affinities by using the Fold-X force field. Proc. Natl. Acad. Sci. USA 102, 10147–10152 (2005).
van der Sloot, A.M. et al. Designed tumor necrosis factor-related apoptosis-inducing ligand variants initiating apoptosis exclusively via the DR5 receptor. Proc. Natl. Acad. Sci. USA 103, 8634–8639 (2006).
Kölsch, V., Seher, T., Fernandez-Ballester, G.J., Serrano, L. & Leptin, M. Control of Drosophila gastrulation by apical localization of adherens junctions and RhoGEF2. Science 315, 384–386 (2007).
Li, X. et al. Deletions of the Aequorea victoria green fluorescent protein define the minimal domain required for fluorescence. J. Biol. Chem. 272, 28545–28549 (1997).
Miller, D.G., Petek, L.M. & Russell, D.W. Human gene targeting by adeno-associated virus vectors is enhanced by DNA double-strand breaks. Mol. Cell. Biol. 23, 3550–3557 (2003).
Sargent, R.G., Brenneman, M.A. & Wilson, J.H. Repair of site-specific double-strand breaks in a mammalian chromosome by homologous and illegitimate recombination. Mol. Cell. Biol. 17, 267–277 (1997).
Porteus, M.H., Cathomen, T., Weitzman, M.D. & Baltimore, D. Efficient gene targeting mediated by adeno-associated virus and DNA double-strand breaks. Mol. Cell. Biol. 23, 3558–3565 (2003).
Smih, F., Rouet, P., Romanienko, P.J. & Jasin, M. Double-strand breaks at the target locus stimulate gene targeting in embryonic stem cells. Nucleic Acids Res. 23, 5012–5019 (1995).
Rogakou, E.P., Boon, C., Redon, C. & Bonner, W.M. Megabase chromatin domains involved in DNA double-strand breaks in vivo. J. Cell Biol. 146, 905–916 (1999).
Rogakou, E.P., Pilch, D.R., Orr, A.H., Ivanova, V.S. & Bonner, W.M. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J. Biol. Chem. 273, 5858–5868 (1998).
Banath, J.P. & Olive, P.L. Expression of phosphorylated histone H2AX as a surrogate of cell killing by drugs that create DNA double-strand breaks. Cancer Res. 63, 4347–4350 (2003).
Catto, L.E., Ganguly, S., Milsom, S.E., Welsh, A.J. & Halford, S.E. Protein assembly and DNA looping by the FokI restriction endonuclease. Nucleic Acids Res. 34, 1711–1720 (2006).
Beumer, K., Bhattacharyya, G., Bibikova, M., Trautman, J.K. & Carroll, D. Efficient gene targeting in Drosophila with zinc-finger nucleases. Genetics 172, 2391–2403 (2006).
Guerois, R., Nielsen, J.E. & Serrano, L. Predicting changes in the stability of proteins and protein complexes: a study of more than 1000 mutations. J. Mol. Biol. 320, 369–387 (2002).
Fernandez-Ballester, G. & Serrano, L. Prediction of protein-protein interaction based on structure. Methods Mol. Biol. 340, 207–234 (2006).
Kiel, C. & Serrano, L. The ubiquitin domain superfold: structure-based sequence alignments and characterization of binding epitopes. J. Mol. Biol. 355, 821–844 (2006).
Vijayakumar, M. et al. Electrostatic enhancement of diffusion-controlled protein-protein association: comparison of theory and experiment on barnase and barstar. J. Mol. Biol. 278, 1015–1024 (1998).
Cathomen, T. & Weitzman, M.D. A functional complex of adenovirus proteins E1B–55kDa and E4orf6 is necessary to modulate the expression level of p53 but not its transcriptional activity. J. Virol. 74, 11407–11412 (2000).
Han, J., Hendzel, M.J. & Allalunis-Turner, J. Quantitative analysis reveals asynchronous and more than double-strand break-associated histone H2AX phosphorylation after exposure to ionizing radiation. Radiat. Res. 165, 283–292 (2006).
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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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.
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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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DOI: https://doi.org/10.1038/nbt1317
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