Improved cell-penetrating zinc-finger nuclease proteins for precision genome engineering

Jia Liu; Thomas Gaj; Mark C Wallen; Carlos F Barbas 3rd

doi:10.1038/mtna.2015.6

Improved cell-penetrating zinc-finger nuclease proteins for precision genome engineering

Mol Ther Nucleic Acids. 2015 Mar 10;4(3):e232. doi: 10.1038/mtna.2015.6.

Authors

Jia Liu¹, Thomas Gaj², Mark C Wallen², Carlos F Barbas 3rd³

Affiliations

¹ 1] The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA [2] Department of Chemistry, The Scripps Research Institute, La Jolla, California, USA [3] Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California, USA [4] Shanghai Institute for Advanced Immunochemical Studies (SIAIS), ShanghaiTech University, Shanghai, China.
² 1] The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA [2] Department of Chemistry, The Scripps Research Institute, La Jolla, California, USA [3] Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California, USA.
³ 1] The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA [2] Department of Chemistry, The Scripps Research Institute, La Jolla, California, USA [3] Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California, USA [4] Deceased.

Abstract

Safe, efficient, and broadly applicable methods for delivering site-specific nucleases into cells are needed in order for targeted genome editing to reach its full potential for basic research and medicine. We previously reported that zinc-finger nuclease (ZFN) proteins have the innate capacity to cross cell membranes and induce genome modification via their direct application to human cells. Here, we show that incorporation of tandem nuclear localization signal (NLS) repeats into the ZFN protein backbone enhances cell permeability nearly 13-fold and that single administration of multi-NLS ZFN proteins leads to genome modification rates of up to 26% in CD4(+) T cells and 17% in CD34(+) hematopoietic stem/progenitor cells. In addition, we show that multi-NLS ZFN proteins attenuate off-target effects and that codelivery of ZFN protein pairs facilitates dual gene modification frequencies of 20-30% in CD4(+) T cells. These results illustrate the applicability of ZFN protein delivery for precision genome engineering.

Abstract

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