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Nat Biotechnol. 2015 Jan;33(1):73-80. doi: 10.1038/nbt.3081. Epub 2014 Oct 30.

Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo.

Author information

1
1] Department of Chemistry &Chemical Biology, Harvard University, Cambridge, Massachusetts, USA. [2] Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA.
2
1] Department of Otology and Laryngology, Harvard Medical School, Boston, Massachusetts, USA. [2] Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, USA. [3] Department of Otology and Skull Base Surgery, Eye, Ear, Nose and Throat Hospital, Shanghai Medical College, Fudan University, Shanghai, China. [4] Key Laboratory of Health Ministry for Hearing Medicine, Shanghai, China.
3
1] Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, Massachusetts, USA. [2] Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts, USA. [3] Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA. [4] Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA.
4
1] Department of Otology and Laryngology, Harvard Medical School, Boston, Massachusetts, USA. [2] Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, USA.

Abstract

Efficient intracellular delivery of proteins is needed to fully realize the potential of protein therapeutics. Current methods of protein delivery commonly suffer from low tolerance for serum, poor endosomal escape and limited in vivo efficacy. Here we report that common cationic lipid nucleic acid transfection reagents can potently deliver proteins that are fused to negatively supercharged proteins, that contain natural anionic domains or that natively bind to anionic nucleic acids. This approach mediates the potent delivery of nM concentrations of Cre recombinase, TALE- and Cas9-based transcription activators, and Cas9:sgRNA nuclease complexes into cultured human cells in media containing 10% serum. Delivery of unmodified Cas9:sgRNA complexes resulted in up to 80% genome modification with substantially higher specificity compared to DNA transfection. This approach also mediated efficient delivery of Cre recombinase and Cas9:sgRNA complexes into the mouse inner ear in vivo, achieving 90% Cre-mediated recombination and 20% Cas9-mediated genome modification in hair cells.

PMID:
25357182
PMCID:
PMC4289409
DOI:
10.1038/nbt.3081
[Indexed for MEDLINE]
Free PMC Article

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