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A multifunctional AAV–CRISPR–Cas9 and its host response

Nature Methods volume 13, pages 868874 (2016) | Download Citation

Abstract

CRISPR–Cas9 delivery by adeno-associated virus (AAV) holds promise for gene therapy but faces critical barriers on account of its potential immunogenicity and limited payload capacity. Here, we demonstrate genome engineering in postnatal mice using AAV–split-Cas9, a multifunctional platform customizable for genome editing, transcriptional regulation, and other previously impracticable applications of AAV–CRISPR–Cas9. We identify crucial parameters that impact efficacy and clinical translation of our platform, including viral biodistribution, editing efficiencies in various organs, antigenicity, immunological reactions, and physiological outcomes. These results reveal that AAV–CRISPR–Cas9 evokes host responses with distinct cellular and molecular signatures, but unlike alternative delivery methods, does not induce extensive cellular damage in vivo. Our study provides a foundation for developing effective genome therapeutics.

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Acknowledgements

We thank R. Chari, H. Lee, D. Mandell, R. Kalhor, A. Chavez, S. Bryne, S. Shipman, V. Busskamp, K. Esvelt, L. Gu, N. Eroshenko, J. Aach, Y. Mayshar, B. Stranges, B. Bauer, K. Hsu, T.G. Tan, A. Castiglioni, T. Serwold, and L. Vandenberghe for discussions; and J. Goldstein for technical assistance. W.L.C. is supported by the National Science Scholarship from the Agency for Science, Technology and Research (A*STAR), Singapore. M.T. is an Albert J. Ryan fellow. This work was supported in part by grants from the Howard Hughes Medical Institute and NIH (UO1 HL100402 and PN2 EY018244 to A.J.W.; and P50 HG005550 to G.M.C.).

Author information

Author notes

    • Prashant Mali
    •  & Elizabeth Y Wu

    Present addresses: Department of Bioengineering, University of California San Diego, La Jolla, California, USA (P.M.) and RaNA Therapeutics, Cambridge, Massachusetts, USA (E.Y.W.).

    • Wei Leong Chew
    •  & Mohammadsharif Tabebordbar

    These authors contributed equally to this work.

Affiliations

  1. Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.

    • Wei Leong Chew
    • , Prashant Mali
    • , Alex H M Ng
    •  & George M Church
  2. Biological and Biomedical Sciences Program, Harvard Medical School, Boston, Massachusetts, USA.

    • Wei Leong Chew
    •  & Mohammadsharif Tabebordbar
  3. Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Harvard University, Cambridge, Massachusetts, USA.

    • Mohammadsharif Tabebordbar
    • , Jason K W Cheng
    • , Elizabeth Y Wu
    • , Kexian Zhu
    •  & Amy J Wagers
  4. Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA.

    • Alex H M Ng
  5. Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA.

    • Kexian Zhu
  6. Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts, USA.

    • George M Church

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Contributions

W.L.C., M.T., A.J.W., and G.M.C. conceived of and designed the study and interpreted results. W.L.C. conducted in vitro experiments, viral production, mouse handling, genotyping, qPCR, western blot, TCR-β clonotyping, epitope mapping, fluorescent immunoassay, histology, immunostaining, microscopy, mRNA sequencing, and data analyses. M.T. conducted mouse handling, intramuscular electroporation, single myofiber isolation, FACS and its analysis, ELISA and its analysis, histology, and immunostaining. P.M. assisted in initial in vitro CRISPR–Cas9 optimization. J.K.W.C. and E.Y.W. conducted mouse handling, histology, and immunostaining. A.H.M.N. conducted mRNA sequencing and its analysis. K.Z. conducted mouse handling. W.L.C. wrote the manuscript with input from the other authors. G.M.C. and A.J.W. supervised the project and edited the manuscript.

Competing interests

W.L.C., M.T., A.J.W., and G.M.C. have filed for patent applications regarding in vivo genetic modifications (PCT/US2015/063181 and WO/2016/089866). W.L.C. and G.M.C. have filed for patent applications regarding split-Cas9 (PCT/US2016/012570 and WO/2016/112242) and AAV-CRISPR-Cas9. M.T. is a current employee of Editas Medicine. G.M.C. is a founder of Editas Medicine and has equity in Caribou/Intellia (for full disclosure list, see http://arep.med.harvard.edu/gmc/tech.html).

Corresponding authors

Correspondence to Amy J Wagers or George M Church.

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    Supplementary Text and Figures

    Supplementary Figures 1–14, Supplementary Table 4, Supplementary Note and Supplementary Sequences.

Excel files

  1. 1.

    Supplementary Table 1

    Total-mRNA sequencing dataset.

  2. 2.

    Supplementary Table 2

    Epitope mapping dataset.

  3. 3.

    Supplementary Table 3

    Admixture transcriptomes deconvolution dataset.

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DOI

https://doi.org/10.1038/nmeth.3993

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