Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA)

Abstract

Engineered zinc-finger nucleases (ZFNs) enable targeted genome modification. Here we describe context-dependent assembly (CoDA), a platform for engineering ZFNs using only standard cloning techniques or custom DNA synthesis. Using CoDA-generated ZFNs, we rapidly altered 20 genes in Danio rerio, Arabidopsis thaliana and Glycine max. The simplicity and efficacy of CoDA will enable broad adoption of ZFN technology and make possible large-scale projects focused on multigene pathways or genome-wide alterations.

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Figure 1: Schematic overview of CoDA.

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Acknowledgements

This work was supported by US National Institutes of Health grants R01 GM088040 (J.K.J. and R.T.P.), R01 GM069906 (J.K.J.), R24 GM078369 (J.K.J.), R01 CA140188 (J.-R.J.Y.), R01 GM081602 (A.J.G.), RC2 MH089956 (A.J.G.), K01 AG031300 (J.-R.J.Y.), K01 AR055619 (D.M.L.) and T32 CA009216 (J.D.S. and J.S.B.); by National Science Foundation grant DBI 0923827 (D.F.V., D.D. and J.K.J.); by the Claflin Distinguished Scholar Award (J.-R.J.Y.); by the Minnesota Soybean Research and Promotion Council (S.J.C. and R.M.S.); and by Alex's Lemonade Stand and the Leukemia Research Foundation (D.M.L.).

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Authors

Contributions

J.D.S. and J.K.J. conceived of the CoDA engineering method; J.D.S., S.J.C., D.M.L., R.M.S., A.J.G., D.F.V., R.T.P., J.-R.J.Y. and J.K.J. designed research; J.D.S., E.J.D., M.J.G., L.C., F.Z., D.C., S.J.C., J.S.B., S.T.-B., Y.Q., C.J.P., E.H., M.L.M. and C.K. performed experiments; J.D.S., D.R. and D.D. identified potential genomic CoDA target sites; and J.D.S., R.M.S., D.F.V., R.T.P., J.-R.J.Y. and J.K.J. wrote the paper.

Corresponding author

Correspondence to J Keith Joung.

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Competing interests

F.Z. and D.F.V. are paid for work performed for Cellectis Plant Sciences.

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Supplementary Figures 1–5, Supplementary Tables 1–5 and Supplementary Discussion (PDF 1275 kb)

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Sander, J., Dahlborg, E., Goodwin, M. et al. Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA). Nat Methods 8, 67–69 (2011). https://doi.org/10.1038/nmeth.1542

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