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

Jia Liu, Thomas Gaj, Mark C. Wallen, Carlos F. Barbas

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Article numbere232
Pages (from-to)e232
JournalMolecular Therapy - Nucleic Acids
Volume4
Issue number3
DOIs
StatePublished - Mar 2015

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Keywords

  • Genome editing
  • Protein delivery
  • Zinc-finger nuclease

ASJC Scopus subject areas

  • Molecular Medicine
  • Drug Discovery

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