Nanoparticle delivery of Cas9 ribonucleoprotein and donor DNA in vivo induces homology-directed DNA repair

Published online:


Clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR associated protein 9 (Cas9)-based therapeutics, especially those that can correct gene mutations via homology-directed repair, have the potential to revolutionize the treatment of genetic diseases. However, it is challenging to develop homology-directed repair-based therapeutics because they require the simultaneous in vivo delivery of Cas9 protein, guide RNA and donor DNA. Here, we demonstrate that a delivery vehicle composed of gold nanoparticles conjugated to DNA and complexed with cationic endosomal disruptive polymers can deliver Cas9 ribonucleoprotein and donor DNA into a wide variety of cell types and efficiently correct the DNA mutation that causes Duchenne muscular dystrophy in mice via local injection, with minimal off-target DNA damage.

  • Subscribe to Nature Biomedical Engineering for full access:



Additional access options:

Already a subscriber?  Log in  now or  Register  for online access.


  1. 1.

    Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–822 (2012).

  2. 2.

    Cong, L. et al. Multiplex genome engineering using CRISPR/Cas system. Science 339, 819–823 (2013).

  3. 3.

    Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).

  4. 4.

    Cho, S. W., Kim, S., Kim, J. M. & Kim, J.-S. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat. Biotechnol. 31, 230–232 (2013).

  5. 5.

    Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–190 (2015).

  6. 6.

    Yin, H. et al. Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo. Nat. Biotechnol. 34, 328–333 (2016).

  7. 7.

    Schumann, K. et al. Generation of knock-in primary human T cells using Cas9 ribonucleoproteins. Proc. Natl Acad. Sci. USA 112, 201512503 (2015).

  8. 8.

    Lin, S., Staahl, B., Alla, R. K. & Doudna, J. A. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. Elife 3, 1–13 (2014).

  9. 9.

    Tabebordbar, M. et al. In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science 351, 407–411 (2016).

  10. 10.

    Nelson, C. E. et al. In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science 351, 403–407 (2016).

  11. 11.

    Long, C. et al. Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy. Science 351, 400–403 (2016).

  12. 12.

    Dever, D. P. et al. CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells. Nature 539, 384–389 (2016).

  13. 13.

    Yin, H. et al. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat. Biotechnol. 32, 551–553 (2014).

  14. 14.

    Lu, Q. L. et al. Systemic delivery of antisense oligoribonucleotide restores dystrophin expression in body-wide skeletal muscles. Proc. Natl Acad. Sci. USA 102, 198–203 (2005).

  15. 15.

    Sun, W. et al. Self-assembled DNA nanoclews for the efficient delivery of CRISPR-Cas9 for genome editing. Angew. Chem. Int. Ed. Engl. 54, 12029–12033 (2015).

  16. 16.

    Kim, S., Kim, D., Cho, S. W., Kim, J. & Kim, J. S. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 24, 1012–1019 (2014).

  17. 17.

    Ramakrishna, S. et al. Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. Genome Res. 24, 1020–1027 (2014).

  18. 18.

    Yu, X. et al. Improved delivery of Cas9 protein/gRNA complexes using lipofectamine CRISPRMAX. Biotechnol. Lett. 38, 919–929 (2016).

  19. 19.

    Miyata, K. et al. Polyplexes from poly(aspartamide) bearing 1,2-diaminoethane side chains induce pH-selective, endosomal membrane destabilization with amplified transfection and negligible cytotoxicity. J. Am. Chem. Soc. 130, 16287–16294 (2008).

  20. 20.

    Kim, H. J. et al. Introduction of stearoyl moieties into a biocompatible cationic polyaspartamide derivative, PAsp(DET), with endosomal escaping function for enhanced siRNA-mediated gene knockdown. J. Control. Release 145, 141–148 (2010).

  21. 21.

    Ding, Y. et al. Gold nanoparticles for nucleic acid delivery. Mol. Ther. 22, 1075–1083 (2014).

  22. 22.

    Singh, D., Sternberg, S. H., Fei, J., Ha, T. & Doudna, J. A. Real-time observation of DNA recognition and rejection by the RNA-guided endonuclease Cas9. Nat. Commun. 7, 1–8 (2016).

  23. 23.

    Sternberg, S. H., Redding, S., Jinek, M., Greene, E. C. & Doudna, J. A. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507, 62–67 (2014).

  24. 24.

    Walkey, C. D. et al. Protein corona fingerprinting predicts the cellular interaction of gold and silver nanoparticles. ACS Nano 8, 2439–2455 (2014).

  25. 25.

    Liu, J. & Peng, Q. Protein-gold nanoparticle interactions and their possible impact on biomedical applications. Acta Biomater. 55, 13–27 (2017).

  26. 26.

    Chithrani, B. D., Ghazani, A. A. & Chan, W. C. W. Determining the size and shape dependence of gold nanoparticle uptake into mammalian cells. Nano Lett. 6, 662–668 (2006).

  27. 27.

    Chou, L. Y. T., Zagorovsky, K. & Chan, W. C. W. DNA assembly of nanoparticle superstructures for controlled biological delivery and elimination. Nat. Nanotechnol. 9, 148–155 (2014).

  28. 28.

    Rouge, J. L., Hao, L., Wu, X. A., Briley, W. E. & Mirkin, C. A. Spherical nucleic acids as a divergent platform for synthesizing RNA–nanoparticle conjugates through enzymatic ligation. ACS Nano 8, 8837–8843 (2014).

  29. 29.

    Smith, R. C., Riollano, M., Leung, A. & Hammond, P. T. Layer-by-layer platform technology for small-molecule delivery. Angew. Chem. Int. Ed. Engl. 48, 8974–8977 (2009).

  30. 30.

    Fu, Y., Sander, J. D., Reyon, D., Cascio, V. M. & Joung, J. K. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat. Biotechnol. 32, 279–284 (2014).

  31. 31.

    Richardson, C. D., Ray, G. J., DeWitt, M. A., Curie, G. L. & Corn, J. E. Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA. Nat. Biotechnol. 34, 339–344 (2016).

  32. 32.

    Lee, K. et al. Synthetically modified guide RNA and donor DNA are a versatile platform for CRISPR-Cas9 engineering. Elife 6, e25312 (2017).

  33. 33.

    Yang, L. et al. Optimization of scarless human stem cell genome editing. Nucleic Acids Res. 41, 9049–9061 (2013).

  34. 34.

    Long, C. et al. Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA. Science 345, 1184–1188 (2014).

  35. 35.

    Zuris, J. A. et al. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat. Biotechnol. 33, 73–80 (2014).

  36. 36.

    Hegde, M. R. et al. Microarray-based mutation detection in the dystrophin gene. Hum. Mutat. 29, 1091–1099 (2008).

  37. 37.

    Nakamura, A. & Takeda, S. Exon-skipping therapy for Duchenne muscular dystrophy. Lancet 378, 546–547 (2011).

  38. 38.

    Mendell, J. R. et al. Eteplirsen for the treatment of Duchenne muscular dystrophy. Ann. Neurol. 74, 637–647 (2013).

  39. 39.

    Kornegay, J. N. et al. Widespread muscle expression of an AAV9 human mini-dystrophin vector after intravenous injection in neonatal dystrophin-deficient dogs. Mol. Ther. 18, 1501–1508 (2010).

  40. 40.

    Xu, L. et al. CRISPR-mediated genome editing restores dystrophin expression and function in mdx mice. Mol. Ther. 24, 564–569 (2016).

  41. 41.

    Pavlath, G. K. & Horsley, V. Cell fusion in skeletal muscle—central role of NFATC2 in regulating muscle cell size. Cell Cycle 2, 420–423 (2003).

  42. 42.

    Sy, J. C. et al. Sustained release of a p38 inhibitor from non-inflammatory microspheres inhibits cardiac dysfunction. Nat. Mater. 863–869 (2008).

Download references


This work was supported by grants from the National Institutes of Health (U01 268201000043C-0-0-1 and R56 AI107116-01 to I.C. as well as grants from Calico, Roger’s and the Strategies for Engineered Negligible Senescence Research Foundation to I.C. This work was also supported by the W. M. Keck Foundation, Moore Foundation, Li Ka Shing Foundation and Center of Innovation programme of the Japan Science and Technology Agency. K.L. is a Siebel Fellow of the Siebel Scholars Foundation. F.J. is a Merck Fellow of the Damon Runyon Cancer Research Foundation (DRG-2201-14). M.A.D. is a California Institute for Regenerative Medicine (CIRM) post-doctoral fellow and is supported by CIRM training grant TG2-01164. J.A.D is a Howard Hughes Medical Institute Investigator. We thank M. West at the CIRM/QB3 Shared Stem Cell Facility and H. Nolla and T. Shovha at the Berkeley FACS facility for technical assistance, as well as D. Schaffer, L. S. Qi, B. Staahl, S. Lin and S. Yang for advice and technical support. This work used the Vincent J. Coates Genomics Sequencing Laboratory at the University of California, Berkeley, supported by National Institutes of Health S10 Instrumentation Grants S10RR029668 and S10RR027303.

Author information

Author notes

  1. Kunwoo Lee, Michael Conboy and Hyo Min Park contributed equally to this work.


  1. GenEdit, Berkeley, CA, 94720-0001, USA

    • Kunwoo Lee
    • , Hyo Min Park
    • , Vanessa A. Mackley
    •  & Hui Liu
  2. Department of Bioengineering, University of California, Berkeley, Berkeley, CA, 94720, USA

    • Michael Conboy
    • , Hyun Jin Kim
    • , Vanessa A. Mackley
    • , Colin Skinner
    • , Tamanna Shobha
    • , Melod Mehdipour
    • , Wen-chin Huang
    • , Freeman Lan
    • , Song Li
    • , Irina Conboy
    •  & Niren Murthy
  3. Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, 94720, USA

    • Fuguo Jiang
    • , Mark A. Dewitt
    • , Kevin Chang
    • , Anirudh Rao
    • , Nicolas L. Bray
    • , Jacob E. Corn
    •  & Jennifer A. Doudna
  4. Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, 113-0033, Japan

    • Hyun Jin Kim
    •  & Kazunori Kataoka
  5. Department of Materials Engineering, Graduate School of Engineering, The University of Tokyo, Tokyo, 113-0033, Japan

    • Hyun Jin Kim
    •  & Kazunori Kataoka
  6. Innovative Genomics Initiative, University of California, Berkeley, Berkeley, CA, 94720, USA

    • Mark A. Dewitt
    • , Nicolas L. Bray
    • , Jacob E. Corn
    •  & Jennifer A. Doudna
  7. Innovation Center of NanoMedicine, Institute of Industry Promotion-KAWASAKI, Kawasaki, 210-0821, Japan

    • Kazunori Kataoka
  8. Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA, 94720, USA

    • Jennifer A. Doudna
  9. Department of Chemistry, University of California, Berkeley, Berkeley, CA, 97420-1460, USA

    • Jennifer A. Doudna
  10. Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA

    • Jennifer A. Doudna


  1. Search for Kunwoo Lee in:

  2. Search for Michael Conboy in:

  3. Search for Hyo Min Park in:

  4. Search for Fuguo Jiang in:

  5. Search for Hyun Jin Kim in:

  6. Search for Mark A. Dewitt in:

  7. Search for Vanessa A. Mackley in:

  8. Search for Kevin Chang in:

  9. Search for Anirudh Rao in:

  10. Search for Colin Skinner in:

  11. Search for Tamanna Shobha in:

  12. Search for Melod Mehdipour in:

  13. Search for Hui Liu in:

  14. Search for Wen-chin Huang in:

  15. Search for Freeman Lan in:

  16. Search for Nicolas L. Bray in:

  17. Search for Song Li in:

  18. Search for Jacob E. Corn in:

  19. Search for Kazunori Kataoka in:

  20. Search for Jennifer A. Doudna in:

  21. Search for Irina Conboy in:

  22. Search for Niren Murthy in:


K.L. planned and performed the experiments shown in all the figures, analysed and interpreted the data and wrote the manuscript. M.C. designed, planned and performed the in vivo studies shown in Figs. 5 and 6 and wrote the manuscript. H.M.P. and H.L. performed the in vivo experiments and analysed and interpreted the data. F.J. purified the Cas9 protein and analysed the gel data. H.J.K. and K.K. synthesized the PAsp(DET) polymer, performed the experiment shown in Supplementary Table 1 and contributed to the data interpretation. W.-c.H. and S.L. cultured the stem cells and performed the experiment shown in Supplementary Fig. 7. M.A.D. and J.E.C. generated the BFP-HEK cells and supported the deep sequencing analysis. V.A.M., K.C., H.M.P. and A.R. performed the in vitro and in vivo DNA analysis. C.S., M.M. and T.S. performed the muscle histology studies. F.L. and N.L.B. performed the deep sequencing analysis. J.E.C. and J.A.D. contributed to the design of the studies and data interpretation. I.C. and N.M. planned and integrated the work, interpreted the data and wrote the manuscript.

Competing interests

K.L., H.M.P. and N.M. are co-founders of GenEdit. J.A.D. is a co-founder of Caribou Biosciences, Editas Medicine and Intellia Therapeutics.

Corresponding authors

Correspondence to Irina Conboy or Niren Murthy.

Electronic supplementary material

  1. Supplementary Information

    Supplementary figures, tables and references.

  2. Life Sciences reporting summary