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

Nature Methods volume 8, pages 74–79 (2011)Cite this article

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Abstract

Zinc-finger nucleases (ZFNs) drive efficient genome editing by introducing a double-strand break into the targeted gene. Cleavage is induced when two custom-designed ZFNs heterodimerize upon binding DNA to form a catalytically active nuclease complex. The importance of this dimerization event for subsequent cleavage activity has stimulated efforts to engineer the nuclease interface to prevent undesired homodimerization. Here we report the development and application of a yeast-based selection system designed to functionally interrogate the ZFN dimer interface. We identified critical residues involved in dimerization through the isolation of cold-sensitive nuclease domains. We used these residues to engineer ZFNs that have superior cleavage activity while suppressing homodimerization. The improvements were portable to orthogonal domains, allowing the concomitant and independent cleavage of two loci using two different ZFN pairs. These ZFN architectures provide a general means for obtaining highly efficient and specific genome modification.

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Figure 1: Isolation of FokI cold-sensitive mutants.
Figure 2: Isolated mutations cluster to the FokI dimer interface.
Figure 3: Enhanced activity of the ELD:KKK and ELD:KKR architecture.
Figure 4: Improved activity of new ZFN mutants in primary cells.
Figure 5: Preservation of the obligate heterodimer specificity.

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Acknowledgements

We thank A. Reik for providing critical reagents and assistance with the manuscript, S. Abrahamson for careful reading of the manuscript and E. Lanphier for encouragement and support.

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  1. Sangamo BioSciences, Richmond, California, USA

    Yannick Doyon, Thuy D Vo, Matthew C Mendel, Shon G Greenberg, Jianbin Wang, Danny F Xia, Jeffrey C Miller, Fyodor D Urnov, Philip D Gregory & Michael C Holmes

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  1. Yannick Doyon
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  2. Thuy D Vo
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  3. Matthew C Mendel
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  8. Fyodor D Urnov
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Contributions

Y.D. and S.G.G. isolated the cold-sensitive mutants. Y.D., T.D.V., M.C.M., J.W. and D.F.X. characterized the engineered domains. Y.D., J.C.M. and M.C.H. designed the experiments. Y.D., F.D.U., P.D.G. and M.C.H. wrote the manuscript.

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Correspondence to Yannick Doyon.

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All authors are employees of Sangamo BioSciences.

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Supplementary Figures 1–15, Supplementary Tables 1–2 and Supplementary Notes 1–2 (PDF 5394 kb)

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Doyon, Y., Vo, T., Mendel, M. et al. Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures. Nat Methods 8, 74–79 (2011). https://doi.org/10.1038/nmeth.1539

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