Abstract
Zinc-finger nucleases (ZFNs) have enabled highly efficient gene targeting in multiple cell types and organisms. Here we describe methods for using simple ssDNA oligonucleotides in tandem with ZFNs to efficiently produce human cell lines with three distinct genetic outcomes: (i) targeted point mutation, (ii) targeted genomic deletion of up to 100 kb and (iii) targeted insertion of small genetic elements concomitant with large genomic deletions.
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Acknowledgements
M.F. acknowledges support from the Danish Cancer Society (R2-A132-02-S2), the Danish National Research Foundation and the Danish Cancer Research Foundation. J.T. acknowledges support from the US National Institutes of Health (GM071434). We thank our colleagues at Sigma-Aldrich and Sangamo Biosciences for helpful advice and discussion; H. Holemon, P. Sullivan and D. Smoller for support; and D. Carroll for helpful comments and discussion.
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F.C., S.M.P.-M., Y.H., M.G. and K.D. performed experiments. G.D.D., F.C., S.M.P.-M. and M.F. designed experiments. G.D.D., T.N.C., M.F., F.C., S.M.P.-M. and J.T. wrote the paper.
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F.C., S.M.P.-M., Y.H., M.G., T.N.C. and G.D.D. are employees of Sigma-Aldrich Corp.
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Chen, F., Pruett-Miller, S., Huang, Y. et al. High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases. Nat Methods 8, 753–755 (2011). https://doi.org/10.1038/nmeth.1653
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DOI: https://doi.org/10.1038/nmeth.1653
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