Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Spatial control of in vivo CRISPR–Cas9 genome editing via nanomagnets

Nature Biomedical Engineering volume 3, pages 126–136 (2019)Cite this article

Subjects

Abstract

The potential of clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR associated protein 9 (Cas9)-based therapeutic genome editing is hampered by difficulties in the control of the in vivo activity of CRISPR–Cas9. To minimize any genotoxicity, precise activation of CRISPR–Cas9 in the target tissue is desirable. Here, we show that, by complexing magnetic nanoparticles with recombinant baculoviral vectors (MNP-BVs), CRISPR–Cas9-mediated genome editing can be activated locally in vivo via a magnetic field. The baculoviral vector was chosen for in vivo gene delivery because of its large loading capacity and ability to locally overcome systemic inactivation by the complement system. We demonstrate that a locally applied magnetic field can enhance the cellular entry of MNP-BVs, thereby avoiding baculoviral vector inactivation and causing a transient transgene expression in the target tissue. Because baculoviral vectors are inactivated elsewhere, gene delivery and in vivo genome editing via MNP-BVs are tissue specific.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Nanomagnets improve endocytosis of baculoviral vector.
Fig. 2: Nanomagnets improve baculoviral vector-mediated transgene expression in vitro.
Fig. 3: Nanomagnets help baculoviral vector overcome serum inactivation in vitro.
Fig. 4: On and off switching of genome editing by the magnetic field and complement system.
Fig. 5: Magnetic-field-enhanced transgene expression and genome editing in subcutaneous tumours.
Fig. 6: A magnetic field enables liver-specific transgene expression and genome editing in vivo via systemic injection.

Data availability

The authors declare that all data supporting the findings of this study are available within the paper and its Supplementary Information. The raw datasets are available from the corresponding author upon reasonable request. The custom script used to analyse the indels in the NGS data is available at https://github.com/piyuranjan/NucleaseIndelActivityScript.

References

  1. Sander, J. D. & Joung, J. K. CRISPR–Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 32, 347–355 (2014).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  4. Swiech, L. et al. In vivo interrogation of gene function in the mammalian brain using CRISPR–Cas9. Nat. Biotechnol. 33, 102–106 (2015).

    Article  CAS  Google Scholar 

  5. Cox, D. B., Platt, R. J. & Zhang, F. Therapeutic genome editing: prospects and challenges. Nat. Med. 21, 121–131 (2015).

    Article  CAS  Google Scholar 

  6. Liao, H. K. et al. Use of the CRISPR–Cas9 system as an intracellular defense against HIV-1 infection in human cells. Nat. Commun. 6, 6413 (2015).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  8. 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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  10. Lin, Y. N. et al. CRISPR–Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences. Nucleic Acids Res. 42, 7473–7485 (2014).

    Article  CAS  Google Scholar 

  11. Cradick, T. J., Fine, E. J., Antico, C. J. & Bao, G. CRISPR/Cas9 systems targeting β-globin and CCR5 genes have substantial off-target activity. Nucleic Acids Res. 41, 9584–9592 (2013).

    Article  CAS  Google Scholar 

  12. Fu, Y. et al. High-frequency off-target mutagenesis induced by CRISPR–Cas nucleases in human cells. Nat. Biotechnol. 31, 822–826 (2013).

    Article  CAS  Google Scholar 

  13. Hsu, P. D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013).

    Article  CAS  Google Scholar 

  14. Lee, C. M., Cradick, T. J., Fine, E. J. & Bao, G. Nuclease target site selection for maximizing on-target activity and minimizing off-target effects in genome editing. Mol. Ther. 24, 475–487 (2016).

    Article  CAS  Google Scholar 

  15. Dow, L. E. et al. Inducible in vivo genome editing with CRISPR–Cas9. Nat. Biotechnol. 33, 390–394 (2015).

    Article  CAS  Google Scholar 

  16. Nihongaki, Y., Kawano, F., Nakajima, T. & Sato, M. Photoactivatable CRISPR–Cas9 for optogenetic genome editing. Nat. Biotechnol. 33, 755–760 (2015).

    Article  CAS  Google Scholar 

  17. Zincarelli, C., Soltys, S., Rengo, G. & Rabinowitz, J. E. Analysis of AAV serotypes 1–9 mediated gene expression and tropism in mice after systemic injection. Mol. Ther. 16, 1073–1080 (2008).

    Article  CAS  Google Scholar 

  18. 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).

    Article  CAS  Google Scholar 

  19. Wang, Y. et al. Systemic dissemination of viral vectors during intratumoral injection. Mol. Cancer Ther. 2, 1233–1242 (2003).

    CAS  PubMed  Google Scholar 

  20. Stanley, S. A., Sauer, J., Kane, R. S., Dordick, J. S. & Friedman, J. M. Remote regulation of glucose homeostasis in mice using genetically encoded nanoparticles. Nat. Med. 21, 92–98 (2015).

    Article  CAS  Google Scholar 

  21. Mannix, R. J. et al. Nanomagnetic actuation of receptor-mediated signal transduction. Nat. Nanotech. 3, 36–40 (2008).

    Article  CAS  Google Scholar 

  22. Wheeler, M. A. et al. Genetically targeted magnetic control of the nervous system. Nat. Neurosci. 19, 756–761 (2016).

    Article  CAS  Google Scholar 

  23. Qiu, Y. et al. Magnetic forces enable controlled drug delivery by disrupting endothelial cell–cell junctions. Nat. Commun. 8, 15594 (2017).

    Article  CAS  Google Scholar 

  24. Sammet, S. Magnetic resonance safety. Abdom. Radiol. 41, 444–451 (2016).

    Article  Google Scholar 

  25. Airenne, K. J. et al. Baculovirus: an insect-derived vector for diverse gene transfer applications. Mol. Ther. 21, 739–749 (2013).

    Article  CAS  Google Scholar 

  26. Mansouri, M. et al. Highly efficient baculovirus-mediated multigene delivery in primary cells. Nat. Commun. 7, 11529 (2016).

    Article  CAS  Google Scholar 

  27. Chen, C. Y., Lin, C. Y., Chen, G. Y. & Hu, Y. C. Baculovirus as a gene delivery vector: recent understandings of molecular alterations in transduced cells and latest applications. Biotechnol. Adv. 29, 618–631 (2011).

    Article  CAS  Google Scholar 

  28. Kost, T. A., Condreay, J. P. & Jarvis, D. L. Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nat. Biotechnol. 23, 567–575 (2005).

    Article  CAS  Google Scholar 

  29. Hindriksen, S. et al. Baculoviral delivery of CRISPR–Cas9 facilitates efficient genome editing in human cells. PLoS ONE 12, e0179514 (2017).

    Article  Google Scholar 

  30. Hofmann, C. & Strauss, M. Baculovirus-mediated gene transfer in the presence of human serum or blood facilitated by inhibition of the complement system. Gene Ther. 5, 531–536 (1998).

    Article  CAS  Google Scholar 

  31. Strauss, R. et al. Baculovirus-based vaccination vectors allow for efficient induction of immune responses against Plasmodium falciparum circumsporozoite protein. Mol. Ther. 15, 193–202 (2007).

    Article  CAS  Google Scholar 

  32. Swift, S. L. et al. Evaluating baculovirus as a vector for human prostate cancer gene therapy. PLoS ONE 8, e65557 (2013).

    Article  CAS  Google Scholar 

  33. Wu, C. et al. Combinatorial control of suicide gene expression by tissue-specific promoter and microRNA regulation for cancer therapy. Mol. Ther. 17, 2058–2066 (2009).

    Article  CAS  Google Scholar 

  34. Haeseleer, F., Imanishi, Y., Saperstein, D. A. & Palczewski, K. Gene transfer mediated by recombinant baculovirus into mouse eye. Invest. Ophthalmol. Vis. Sci. 42, 3294–3300 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Kaikkonen, M. U., Maatta, A. I., Yla-Herttuala, S. & Airenne, K. J. Screening of complement inhibitors: shielded baculoviruses increase the safety and efficacy of gene delivery. Mol. Ther. 18, 987–992 (2010).

    Article  CAS  Google Scholar 

  36. Raty, J. K. et al. Enhanced gene delivery by avidin-displaying baculovirus. Mol. Ther. 9, 282–291 (2004).

    Article  CAS  Google Scholar 

  37. Sun, S. et al. Monodisperse MFe2O4 (M = Fe, Co, Mn) nanoparticles. J. Am. Chem. Soc. 126, 273–279 (2004).

    Article  CAS  Google Scholar 

  38. Tong, S., Hou, S., Ren, B., Zheng, Z. & Bao, G. Self-assembly of phospholipid-PEG coating on nanoparticles through dual solvent exchange. Nano Lett. 11, 3720–3726 (2011).

    Article  CAS  Google Scholar 

  39. Torchilin, V. P. TAT peptide-mediated intracellular delivery of pharmaceutical nanocarriers. Adv. Drug Deliv. Rev. 60, 548–558 (2008).

    Article  CAS  Google Scholar 

  40. Small, D. A. & Moore, N. F. Measurement of surface charge of baculovirus polyhedra. Appl. Environ. Microbiol. 53, 598–602 (1987).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Boyce, F. M. & Bucher, N. L. R. Baculovirus-mediated gene transfer into mammalian cells. Proc. Natl Acad. Sci. USA 93, 2348–2352 (1996).

    Article  CAS  Google Scholar 

  42. Ohkawa, T., Volkman, L. E. & Welch, M. D. Actin-based motility drives baculovirus transit to the nucleus and cell surface. J. Cell Biol. 190, 187–195 (2010).

    Article  CAS  Google Scholar 

  43. Matilainen, H. et al. Baculovirus entry into human hepatoma cells. J. Virol. 79, 15452–15459 (2005).

    Article  CAS  Google Scholar 

  44. Kataoka, C. et al. Baculovirus GP64-mediated entry into mammalian cells. J. Virol. 86, 2610–2620 (2012).

    Article  CAS  Google Scholar 

  45. Romet-Lemonne, G. & Jegou, A. Mechanotransduction down to individual actin filaments. Eur. J. Cell Biol. 92, 333–338 (2013).

    Article  CAS  Google Scholar 

  46. Shen, H., Tong, S., Bao, G. & Wang, B. Structural responses of cells to intracellular magnetic force induced by superparamagnetic iron oxide nanoparticles. Phys. Chem. Chem. Phys. 16, 1914–1920 (2014).

    Article  CAS  Google Scholar 

  47. Yuan, F. et al. Vascular permeability in a human tumor xenograft: molecular size dependence and cutoff size. Cancer Res. 55, 3752–3756 (1995).

    CAS  PubMed  Google Scholar 

  48. Danquah, J. O., Botchway, S., Jeshtadi, A. & King, L. A. Direct interaction of baculovirus capsid proteins VP39 and EXON0 with kinesin-1 in insect cells determined by fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy. J. Virol. 86, 844–853 (2012).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank L. Volkman and T. Ohkawa for kindly providing the anti-vp39 antibody, and T. Davis, L. Hong and A. Ray for assistance. This work was supported by the National Institutes of Health through a Nanomedicine Development Center Award (PN2EY018244 to G.B.) and the Cancer Prevention and Research Institute of Texas (RR140081 and RR170721 to G.B.).

Author information

Author notes

  1. These authors contributed equally: Haibao Zhu, Linlin Zhang, Sheng Tong.

Authors and Affiliations

  1. Department of Bioengineering, Rice University, Houston, TX, USA

    Haibao Zhu, Linlin Zhang, Sheng Tong, Ciaran M. Lee, Harshavardhan Deshmukh & Gang Bao

Authors
  1. Haibao Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Linlin Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sheng Tong
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ciaran M. Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Harshavardhan Deshmukh
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Gang Bao
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

H.Z., S.T. and G.B. conceived the idea, designed the study and wrote the manuscript. H.Z., S.T., L.Z. and H.D. performed the experiments and data analysis. C.M.L. helped with the CRISPR guide RNA design and performed the NGS analysis.

Corresponding author

Correspondence to Gang Bao.

Ethics declarations

Competing interests

H.Z., S.T. and G.B. filed a US patent application (US20170239370A1) based on the results presented in this paper.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary figures and tables.

Reporting Summary

Supplementary Video 1

Fluorescence microscopy of cells incubated with BV.

Supplementary Video 2

Fluorescence microscopy of cells incubated with MNP-BV.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Zhu, H., Zhang, L., Tong, S. et al. Spatial control of in vivo CRISPR–Cas9 genome editing via nanomagnets. Nat Biomed Eng 3, 126–136 (2019). https://doi.org/10.1038/s41551-018-0318-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41551-018-0318-7

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research