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An optimized two-finger archive for ZFN-mediated gene targeting

Abstract

The widespread use of zinc-finger nucleases (ZFNs) for genome engineering is hampered by the fact that only a subset of sequences can be efficiently recognized using published finger archives. We describe a set of validated two-finger modules that complement existing finger archives and expand the range of ZFN-accessible sequences threefold. Using this archive, we introduced lesions at 9 of 11 target sites in the zebrafish genome.

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Figure 1: Selection and characterization of two-finger modules recognizing GANNCG sequences.

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Acknowledgements

This research was supported by the US National Institutes of Health R01GM068110 (S.A.W.), R24GM078369, R01HL093766 (N. Lawson & S.A.W.) and R01HG00249 (G.D.S.). We thank N. Lawson and members of his laboratory for their insightful advice and zebrafish husbandry training, and J. Zhu for assistance with website construction.

Author information

Authors and Affiliations

Authors

Contributions

S.A.W. conceived the study; A.G. and A.L.R. carried out the selection experiments. R.G.C. and G.D.S. developed the computational platform for motif analysis. A.L. performed the analysis of ZFN sites in multiple genomes. A.G. and S.A.W. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Scot A Wolfe.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10, Supplementary Tables 3–10, Supplementary Discussion 1–3, Supplementary Methods (PDF 2268 kb)

Supplementary Table 1

List of all two-finger modules obtained after B1H-based selections from the Asn+3F2 library and the His+3F2 library. Recognition helix sequences (–1, 1, 2, 3, 4, 5, 6) for finger 1 and finger 2 for selected two-finger modules are shown for two-finger modules selected for zinc-finger binding sites (gaNNcg where NN represents the 2-bp interface). The amino acid at position 3 of finger 2 can either be Asn or His depending on the zinc finger library used. The selection conditions (3-AT, IPTG and uracil concentrations) are given. For selection of two-finger modules that bind the G-G interface with the Asn+3F2 library, a mutant homeodomain binding site (TAAAGG) was used. (XLS 102 kb)

Supplementary Table 2

List of all two-finger modules and target sites for the assembly of ZFNs. Recognition helix sequences for F1 and F2 of each two-finger module are shown. (XLS 57 kb)

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Gupta, A., Christensen, R., Rayla, A. et al. An optimized two-finger archive for ZFN-mediated gene targeting. Nat Methods 9, 588–590 (2012). https://doi.org/10.1038/nmeth.1994

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