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Reply to “Genome editing with modularly assembled zinc-finger nucleases”

Nature Methods volume 7, pages 91–92 (2010)Cite this article

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Joung et al. reply:

The publications cited by Kim et al.1 describing successful construction of zinc-finger nucleases (ZFNs) by modular assembly only further support our original conclusion that this method has a high failure rate for engineering functional zinc-finger arrays2. Two3,4 of the three reports cited in their Correspondence1 provide data that enable calculation of failure rates for modular assembly. Although it is true that modular assembly yielded ZFNs for ∼25% of the DNA sites targeted, failure rates measured instead by the number of zinc-finger proteins tested are consistent with those reported in our original Correspondence2. For example, at the human CCR5 gene, Kim et al. screened 315 pairs of ZFNs for activity4; this large-scale effort yielded only a small number of functional ZFN pairs (93.3% failure rate for ZFN pairs tested). Similarly, for the tobacco SuRB gene3, we tested 32 zinc-finger arrays in vitro but identified only three with functional activity (91.6% failure rate for zinc-finger arrays tested). These data are consistent with our original predicted failure rates of ∼94% and ∼76% for modularly assembled ZFN pairs and zinc-finger arrays, respectively2. We believe that failure rates measured by numbers of zinc-finger arrays or ZFN pairs tested rather than by numbers of DNA sites targeted are more relevant statistics for potential ZFN users because these influence how many proteins must be modularly assembled and tested for each potential site.

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References

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Acknowledgements

We thank the members of our laboratories for helpful discussions. J.K.J. is supported by US National Institutes of Health (R01GM069906, R01GM088040, RC2HL101553, R24GM078369 and R21HL091808), the Cystic Fibrosis Foundation and the Massachusetts General Hospital Pathology Service. D.F.V. is supported by the US National Science Foundation (DBI 0501678 and MCB 0209818). T.C. is supported by the German Research Foundation (SPP1230–CA311/2), the German Ministry of Education and Research (01GU0618) and the European Commission's 6th and 7th Framework Programmes (ZNIP–037783 and PERSIST–222878).

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Authors and Affiliations

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

    J Keith Joung

  2. Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA

    J Keith Joung

  3. Department of Genetics, Cell Biology and Development, and Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota, USA

    Daniel F Voytas

  4. Department of Experimental Hematology, Hannover Medical School, Hannover, Germany

    Toni Cathomen

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  1. J Keith Joung
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  2. Daniel F Voytas
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  3. Toni Cathomen
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Correspondence to J Keith Joung, Daniel F Voytas or Toni Cathomen.

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Joung, J., Voytas, D. & Cathomen, T. Reply to “Genome editing with modularly assembled zinc-finger nucleases”. Nat Methods 7, 91–92 (2010). https://doi.org/10.1038/nmeth0210-91b

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