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

Nature Biotechnology volume 33, pages 73–80 (2015)Cite this article

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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.

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Figure 1: Strategy for delivering proteins into mammalian cells by fusion or noncovalent complexation with polyanionic macromolecules and complexation with cationic lipids.
Figure 2: Delivery of Cre recombinase to cultured human cells.
Figure 3: Delivery of TALE transcriptional activators into cultured human cells.
Figure 4: Delivery of Cas9:sgRNA, Cas9 D10A nickase, and dCas9-VP64 transcription activators to cultured human cells.
Figure 5: DNA sequence specificity of Cas9-mediated endogenous gene cleavage in cultured human cells by plasmid transfection or by cationic lipid–mediated protein:sgRNA delivery using 1.6 μl RNAiMAX complexed with 100 nM Cas9 and 100 nM sgRNA.
Figure 6: In vivo delivery of Cre recombinase and Cas9:sgRNA complexes to hair cells in the mouse inner ear.

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Acknowledgements

J.A.Z. is a Ruth L. Kirchstein National Research Service Awards Postdoctoral Fellow (F32 GM 106601-2) D.B.T., J.P.G. and J.L.B. were supported by US National Institutes of Health (NIH) R01 GM095501 (to D.R.L.), Defense Advanced Research Projects Agency HR0011-11-2-0003 (to D.R.L.) and N66001-12-C-4207 (to D.R.L.), and the Howard Hughes Medical Institute (HHMI). D.R.L. was supported as a HHMI Investigator. Z.-Y. C. was supported by US National Institutes of Health (R01 DC006908), the Bertarelli Foundation, and the David-Shulsky Foundation. Y.S. was supported by the Frederick and Ines Yeatts Hair Cell Regeneration grant and by The National Nature Science Foundation of China NSFC81300824. J.H.H. was supported by National Science Foundation Graduate Research Fellowship Program (DGE1144152). M.L.M. and J.K.J. were supported by an NIH Director's Pioneer Award (DP1 GM105378). We thank A. Lawson, M. Sonnett, R. Xiao, S. Wang and J. Gehrke for technical assistance. We thank A. Edge, Massachusetts Eye & Ear Infirmary, Boston, for mouse embryonic stem cell (ES) line Tau-GFP and J. Johnson, Southwestern Medical Center, University of Texas, for floxP-tdTomato mice (The Jackson Laboratory).

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Authors and Affiliations

  1. Department of Chemistry & Chemical Biology, Harvard University, Cambridge, Massachusetts, USA

    John A Zuris, David B Thompson, John P Guilinger, Jeffrey L Bessen, Johnny H Hu & David R Liu

  2. Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA

    John A Zuris, David B Thompson, John P Guilinger, Jeffrey L Bessen, Johnny H Hu & David R Liu

  3. Department of Otology and Laryngology, Harvard Medical School, Boston, Massachusetts, USA

    Yilai Shu & Zheng-Yi Chen

  4. Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, USA

    Yilai Shu & Zheng-Yi Chen

  5. Department of Otology and Skull Base Surgery, Eye, Ear, Nose and Throat Hospital, Shanghai Medical College, Fudan University, Shanghai, China

    Yilai Shu

  6. Key Laboratory of Health Ministry for Hearing Medicine, Shanghai, China

    Yilai Shu

  7. Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, Massachusetts, USA

    Morgan L Maeder & J Keith Joung

  8. Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts, USA

    Morgan L Maeder & J Keith Joung

  9. Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA

    Morgan L Maeder & J Keith Joung

  10. Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA

    Morgan L Maeder & J Keith Joung

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  1. John A Zuris
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Contributions

J.A.Z., D.B.T., Y.S., Z.-Y.C. and D.R.L. designed the research and analyzed the data. J.A.Z., D.B.T., Y.S., J.P.G. and J.L.B. generated research materials and performed the experiments. M.L.M. designed and constructed TALEs and dCas9 activator sgRNAs. J.P.G., J.A.Z. and J.H.H. analyzed DNA sequencing data. J.K.J., Z.-Y.C. and D.R.L. supervised the research. All authors wrote the manuscript.

Corresponding author

Correspondence to David R Liu.

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Competing interests

The co-authors have filed a provisional patent application related to this work. J.K.J. and D.R.L. are cofounders of consultants for Editas Medicine, a company that applies genome-editing technologies. J.K.J. is a consultant for Horizon Discovery. J.K.J. has financial interests in Editas Medicine and Transposagen Biopharmaceuticals. J.K.J.'s interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies. M.L.M. is currently an employee of Editas Medicine.

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Zuris, J., Thompson, D., Shu, Y. et al. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotechnol 33, 73–80 (2015). https://doi.org/10.1038/nbt.3081

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