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

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

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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.

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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.

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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.

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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.).

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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

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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.

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Correspondence to Gang Bao.

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Competing interests

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

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Supplementary Information

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Reporting Summary

Supplementary Video 1

Fluorescence microscopy of cells incubated with BV.

Supplementary Video 2

Fluorescence microscopy of cells incubated with MNP-BV.

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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

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