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Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection

Nature Methods volume 8, pages 765–770 (2011)Cite this article

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Abstract

Engineered zinc-finger nucleases (ZFNs) are promising tools for genome manipulation, and determining off-target cleavage sites of these enzymes is of great interest. We developed an in vitro selection method that interrogates 1011 DNA sequences for cleavage by active, dimeric ZFNs. The method revealed hundreds of thousands of DNA sequences, some present in the human genome, that can be cleaved in vitro by two ZFNs: CCR5-224 and VF2468, which target the endogenous human CCR5 and VEGFA genes, respectively. Analysis of identified sites in one cultured human cell line revealed CCR5-224–induced changes at nine off-target loci, though this remains to be tested in other relevant cell types. Similarly, we observed 31 off-target sites cleaved by VF2468 in cultured human cells. Our findings establish an energy compensation model of ZFN specificity in which excess binding energy contributes to off-target ZFN cleavage and suggest strategies for the improvement of future ZFN design.

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Figure 1: In vitro selection for ZFN-mediated cleavage.
Figure 2: DNA cleavage sequence specificity profiles for CCR5-224 and VF2468 ZFNs.
Figure 3: Evidence for a compensation model of ZFN target site recognition.
Figure 4: ZFNs can cleave a large fraction of target sites with three or fewer mutations in vitro.

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References

  1. 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).

    Article  CAS  Google Scholar 

  2. Vanamee, E.S., Santagata, S. & Aggarwal, A.K. FokI requires two specific DNA sites for cleavage. J. Mol. Biol. 309, 69–78 (2001).

    Article  CAS  Google Scholar 

  3. Hockemeyer, D. et al. Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nat. Biotechnol. 27, 851–857 (2009).

    Article  CAS  Google Scholar 

  4. Maeder, M.L. et al. Rapid “open-source” engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol. Cell 31, 294–301 (2008).

    Article  CAS  Google Scholar 

  5. Zou, J. et al. Gene targeting of a disease-related gene in human induced pluripotent stem and embryonic stem cells. Cell Stem Cell 5, 97–110 (2009).

    Article  CAS  Google Scholar 

  6. Perez, E.E. et al. Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nat. Biotechnol. 26, 808–816 (2008).

    Article  CAS  Google Scholar 

  7. Urnov, F.D. et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435, 646–651 (2005).

    Article  CAS  Google Scholar 

  8. Santiago, Y. et al. Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases. Proc. Natl. Acad. Sci. USA 105, 5809–5814 (2008).

    Article  CAS  Google Scholar 

  9. Cui, X. et al. Targeted integration in rat and mouse embryos with zinc-finger nucleases. Nat. Biotechnol. 29, 64–67 (2011).

    Article  CAS  Google Scholar 

  10. Cornu, T.I. et al. DNA-binding specificity is a major determinant of the activity and toxicity of zinc-finger nucleases. Mol. Ther. 16, 352–358 (2008).

    Article  CAS  Google Scholar 

  11. 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).

    Article  CAS  Google Scholar 

  12. Bulyk, M.L., Huang, X., Choo, Y. & Church, G.M. Exploring the DNA-binding specificities of zinc fingers with DNA microarrays. Proc. Natl. Acad. Sci. USA 98, 7158–7163 (2001).

    Article  CAS  Google Scholar 

  13. Meng, X., Thibodeau-Beganny, S., Jiang, T., Joung, J.K. & Wolfe, S.A. Profiling the DNA-binding specificities of engineered Cys2His2 zinc finger domains using a rapid cell-based method. Nucleic Acids Res. 35, e81 (2007).

    Article  Google Scholar 

  14. Wolfe, S.A., Greisman, H.A., Ramm, E.I. & Pabo, C.O. Analysis of zinc fingers optimized via phage display: evaluating the utility of a recognition code. J. Mol. Biol. 285, 1917–1934 (1999).

    Article  CAS  Google Scholar 

  15. Segal, D.J. et al. Evaluation of a modular strategy for the construction of novel polydactyl zinc finger DNA-binding proteins. Biochemistry 42, 2137–2148 (2003).

    Article  CAS  Google Scholar 

  16. Zykovich, A., Korf, I. & Segal, D.J. Bind-n-Seq: high-throughput analysis of in vitro protein-DNA interactions using massively parallel sequencing. Nucleic Acids Res. 37, e151 (2009).

    Article  Google Scholar 

  17. Yanover, C. & Bradley, P. Extensive protein and DNA backbone sampling improves structure-based specificity prediction for C2H2 zinc fingers. Nucleic Acids Res. 39, 4564–4576 (2011).

    Article  CAS  Google Scholar 

  18. 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).

    Article  CAS  Google Scholar 

  19. Bibikova, M., Golic, M., Golic, K.G. & Carroll, D. Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Genetics 161, 1169–1175 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Gupta, A., Meng, X., Zhu, L.J., Lawson, N.D. & Wolfe, S.A. Zinc finger protein-dependent and -independent contributions to the in vivo off-target activity of zinc finger nucleases. Nucleic Acids Res. 39, 381–392 (2011).

    Article  CAS  Google Scholar 

  21. Soldner, F. et al. Generation of isogenic pluripotent stem cells differing exclusively at two early onset Parkinson point mutations. Cell 146, 318–331 (2011).

    Article  CAS  Google Scholar 

  22. Chen, J. et al. Molecular cloning and characterization of a novel human BTB domain-containing gene, BTBD10, which is down-regulated in glioma. Gene 340, 61–69 (2004).

    Article  CAS  Google Scholar 

  23. Wang, X. et al. Glucose metabolism-related protein 1 (GMRP1) regulates pancreatic beta cell proliferation and apoptosis via activation of Akt signalling pathway in rats and mice. Diabetologia 54, 852–863 (2011).

    Article  CAS  Google Scholar 

  24. Nawa, M., Kanekura, K., Hashimoto, Y., Aiso, S. & Matsuoka, M. A novel Akt/PKB-interacting protein promotes cell adhesion and inhibits familial amyotrophic lateral sclerosis-linked mutant SOD1-induced neuronal death via inhibition of PP2A-mediated dephosphorylation of Akt/PKB. Cell Signal. 20, 493–505 (2008).

    Article  CAS  Google Scholar 

  25. Petek, L.M., Russell, D.W. & Miller, D.G. Frequent endonuclease cleavage at off-target locations in vivo. Mol. Ther. 18, 983–986 (2010).

    Article  CAS  Google Scholar 

  26. 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).

    Article  CAS  Google Scholar 

  27. Ramirez, C.L. et al. Unexpected failure rates for modular assembly of engineered zinc fingers. Nat. Methods 5, 374–375 (2008).

    Article  CAS  Google Scholar 

  28. Bibikova, M. et al. Stimulation of homologous recombination through targeted cleavage by chimeric nucleases. Mol. Cell Biol. 21, 289–297 (2001).

    Article  CAS  Google Scholar 

  29. Handel, E.M., Alwin, S. & Cathomen, T. Expanding or restricting the target site repertoire of zinc-finger nucleases: the inter-domain linker as a major determinant of target site selectivity. Mol. Ther. 17, 104–111 (2009).

    Article  Google Scholar 

  30. Miller, J.C. et al. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat. Biotechnol. 25, 778–785 (2007).

    Article  CAS  Google Scholar 

  31. Cradick, T.J., Keck, K., Bradshaw, S., Jamieson, A.C. & McCaffrey, A.P. Zinc-finger nucleases as a novel therapeutic strategy for targeting hepatitis B virus DNAs. Mol. Ther. 18, 947–954 (2010).

    Article  CAS  Google Scholar 

  32. Doyon, Y. et al. Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases. Nat. Biotechnol. 26, 702–708 (2008).

    Article  CAS  Google Scholar 

  33. Rozen, S. & Skaletsky, H. Primer3 on the WWW for general users and for biologist programmers. Methods Mol. Biol. 132, 365–386 (2000).

    CAS  Google Scholar 

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Acknowledgements

This research was supported by US National Institutes of Health (NIH) R01 GM065400 (D.R.L.), Defense Advanced Research Projects Agency HR0011-11-2-0003 (D.R.L.), the Howard Hughes Medical Institute (D.R.L.), NIH R01 GM088040 (J.K.J.), NIH DP1 OD006862 (J.K.J.), and the Jim and Ann Orr Massachusetts General Hospital Research Scholar award (J.K.J.). V.P. was supported by an NIH training grant to the Harvard University Training Program in Molecular, Cellular and Chemical Biology. C.L.R. was supported by a National Science Foundation Graduate Research Fellowship and a Ford Foundation Predoctoral Fellowship. The Massachusetts General Hospital Neuroscience core facility, supported by NIH P30 NS045776, provided qPCR capabilities. We thank J. Carlson, B. Dorr, C. Pattanayak, D. Reyon, J. Sander and D. Thompson for helpful discussions, M. Goodwin for technical assistance, and M. Maeder (Massachusetts General Hospital) for mammalian cell ZFN expression plasmids.

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Authors and Affiliations

  1. Department of Chemistry and Chemical Biology, and Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA

    Vikram Pattanayak & David R Liu

  2. Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA

    Cherie L Ramirez & J Keith Joung

  3. Department of Pathology and Biological and Biomedical Sciences Program, Harvard Medical School, Boston, Massachusetts, USA

    Cherie L Ramirez & J Keith Joung

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  1. Vikram Pattanayak
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Contributions

V.P. and C.L.R. performed the experiments, designed the research, analyzed the data and wrote the manuscript. J.K.J. and D.R.L. designed the research, analyzed the data and wrote the manuscript.

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Correspondence to David R Liu.

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Supplementary Figures 1–15, Supplementary Tables 1–7, Supplementary Notes 1–2, Supplementary Protocols 1–9, Supplementary Data (PDF 5131 kb)

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Pattanayak, V., Ramirez, C., Joung, J. et al. Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection. Nat Methods 8, 765–770 (2011). https://doi.org/10.1038/nmeth.1670

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