Journal
NATURE BIOTECHNOLOGY
Volume 33, Issue 1, Pages 73-80Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nbt.3081
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Funding
- Ruth L. Kirchstein National Research Service Award [F32 GM 106601-2]
- US National Institutes of Health (NIH) [R01 GM095501, R01 DC006908]
- Defense Advanced Research Projects Agency [HR0011-11-2-0003, N66001-12-C-4207]
- Howard Hughes Medical Institute (HHMI)
- Bertarelli Foundation
- David-Shulsky Foundation
- Frederick and Ines Yeatts Hair Cell Regeneration grant
- National Nature Science Foundation of China [NSFC81300824]
- National Science Foundation Graduate Research Fellowship Program [DGE1144152]
- NIH Director's Pioneer Award [DP1 GM105378]
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R01GM095501, F32GM106601] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE ON DEAFNESS AND OTHER COMMUNICATION DISORDERS [R01DC006908] Funding Source: NIH RePORTER
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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.
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