An improved zinc-finger nuclease architecture for highly specific genome editing

Nature Biotechnology volume 25, pages778–785(2007)Cite this article

Abstract

Genome editing driven by zinc-finger nucleases (ZFNs) yields high gene-modification efficiencies (>10%) by introducing a recombinogenic double-strand break into the targeted gene. The cleavage event is induced using two custom-designed ZFNs that heterodimerize upon binding DNA to form a catalytically active nuclease complex. Using the current ZFN architecture, however, cleavage-competent homodimers may also form that can limit safety or efficacy via off-target cleavage. Here we develop an improved ZFN architecture that eliminates this problem. Using structure-based design, we engineer two variant ZFNs that efficiently cleave DNA only when paired as a heterodimer. These ZFNs modify a native endogenous locus as efficiently as the parental architecture, but with a >40-fold reduction in homodimer function and much lower levels of genome-wide cleavage. This architecture provides a general means for improving the specificity of ZFNs as gene modification reagents.

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Figure 1: DNA recognition and cleavage by zinc-finger nucleases (ZFNs).
Figure 2: Development of FokI cleavage domain mutants that function as obligate heterodimers.
Figure 3: Gene editing activity of FokI cleavage domain variants at a native endogenous target.
Figure 4: Preferential heterodimer activity of FokI variants as demonstrated by DNA cleavage in vitro. (a) DNA target fragments.
Figure 5: Reduced DNA damage levels by heterodimer variants.
Figure 6: Heterodimer variants yield reduced DNA damage levels while retaining full activity for targeted gene modification.

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Acknowledgements

We thank Judy Campisi for support and helpful discussions, Yann Jouvenot, Sheldon Augustus, Danny Xia and Lei Zhang for assistance with protocols and constructs, Dana Carroll for comments on the manuscript, Fyodor Urnov for helpful discussions and Edward Lanphier for encouragement and support. This research was supported in part by grant no. 7ONANB4H3006 from the Advanced Technology Program (US Department of Energy).

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Author notes

  1. Christian M Beausejour & Carl O Pabo

    Present address: Present addresses: Département de pharmacologie, Centre de Recherche, CHU Ste-Justine 3175, Côte Ste-Catherine, Montréal, Quebec H3T 1C5, Canada (C.M.B.) and Department of Systems Biology, Harvard Medical School, 200 Longwood Avenue, WAB 536, Boston, Massachusetts 02115, USA (C.O.P.).,

Affiliations

  1. Sangamo BioSciences, Inc., Pt. Richmond Tech Center, 501 Canal Blvd., Suite A100, Richmond, 94804, California, USA

    Jeffrey C Miller, Michael C Holmes, Jianbin Wang, Dmitry Y Guschin, Ya-Li Lee, Igor Rupniewski, Christian M Beausejour, Adam J Waite, Nathaniel S Wang, Kenneth A Kim, Philip D Gregory, Carl O Pabo & Edward J Rebar

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  1. Jeffrey C Miller
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  2. Michael C Holmes
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Contributions

J.C.M and C.O.P. conceived the project; J.C.M., M.C.H., P.D.G., C.O.P. and E.J.R. designed experiments; J.C.M., M.C.H., J.W., D.Y.G., Y.-L.L., I.R., C.M.B., A.J.W., N.S.W and K.A.K. performed the experiments; J.C.M., M.C.H., P.D.G., C.O.P. and E.J.R. wrote the manuscript.

Corresponding author

Correspondence to Edward J Rebar.

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Competing interests

Authors of this work are current or former employees of Sangamo BioSciences, Inc. (C.O.P. was CSO from 2001–2003; he retains a role as chair of the scientific advisory board.)

Supplementary information

Supplementary Fig. 1

In vitro DNA cleavage assay with all four combinations of the “plus” and “minus” cleavage domain variants. (DOC 34 kb)

Supplementary Fig. 2

Sequences of nuclease constructs. (DOC 28 kb)

Supplementary Table 1

Activity data for key constructs tested during our iterative development process. (DOC 94 kb)

Supplementary Methods (DOC 33 kb)

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Miller, J., Holmes, M., Wang, J. et al. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol 25, 778–785 (2007). https://doi.org/10.1038/nbt1319

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