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Targeted gene knockout by direct delivery of zinc-finger nuclease proteins


Zinc-finger nucleases (ZFNs) are versatile reagents that have redefined genome engineering. Realizing the full potential of this technology requires the development of safe and effective methods for delivering ZFNs into cells. We demonstrate the intrinsic cell-penetrating capabilities of the standard ZFN architecture and show that direct delivery of ZFNs as proteins leads to efficient endogenous gene disruption in various mammalian cell types with minimal off-target effects.

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Figure 1: ZFN proteins are cell permeable and induce targeted mutagenesis in human cells.
Figure 2: Modification of endogenous genes by direct delivery of ZFN proteins.

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We thank C. Gersbach for contributing to preliminary studies, P. Ikrenyi for technical assistance and A. Mercer for discussion of the manuscript. This research was supported by US National Institutes of Health (NIH) grants GM065059 and DP1OD006990 and by The Skaggs Institute for Chemical Biology. T.G. was supported by a US National Institute of General Medicine Sciences fellowship (T32GM080209). Y.K. was supported by the Japan Society for the Promotion of Science Research Fellowships for Young Scientists.

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T.G., J.G., Y.K., S.J.S. and C.F.B. designed the research; J.G. and Y.K. purified ZFN proteins; T.G., J.G., Y.K. and S.J.S. performed experiments; T.G., J.G., Y.K., S.J.S. and C.F.B. analyzed data; T.G., S.J.S. and C.F.B. wrote the manuscript.

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Correspondence to Carlos F Barbas III.

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Competing interests

The authors are inventors on wide-ranging patents concerning the development and use of zinc-finger technology (US Patent Nos. 6,140,081, 6,140,466, 6,242,568, 6,610,512, 6,790,941, 7,011,972, 7,067,617, 7,101,972, 7,151,201, 7,329,541, 7,329,728, 7,378,510, 7, 442,784, 7,741,110, 7,781,645 and 7,833,784) and have also submitted an application to the US Patent Office concerning the contents of this manuscript.

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Gaj, T., Guo, J., Kato, Y. et al. Targeted gene knockout by direct delivery of zinc-finger nuclease proteins. Nat Methods 9, 805–807 (2012).

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