The discovery of the highly efficient site-specific nuclease system CRISPR–Cas9 from Streptococcus pyogenes has galvanized the field of gene therapy1,2. The immunogenicity of Cas9 nuclease has been demonstrated in mice3,4. Preexisting immunity against therapeutic gene vectors or their cargo can decrease the efficacy of a potentially curative treatment and may pose significant safety issues3,4,5,6. S. pyogenes is a common cause for infectious diseases in humans, but it remains unclear whether it induces a T cell memory against the Cas9 nuclease7,8. Here, we show the presence of a preexisting ubiquitous effector T cell response directed toward the most widely used Cas9 homolog from S. pyogenes (SpCas9) within healthy humans. We characterize SpCas9-reactive T cells within the CD4/CD8 compartments for multi-effector potency, cytotoxicity, and lineage determination. In-depth analysis of SpCas9-reactive T cells reveals a high frequency of SpCas9-reactive regulatory T cells that can mitigate SpCas9-reactive effector T cell proliferation and function in vitro. Our results shed light on T cell–mediated immunity toward CRISPR-associated nucleases and offer a possible solution to overcome the problem of preexisting immunity.

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Data availability

The underlying data are available from the corresponding author upon reasonable request. TRB sequencing data have been deposited in the immuneACCESS database at https://doi.org/10.21417/B7ZP86 (http://clients.adaptivebiotech.com/pub/wagner-2018-naturemedicine).

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Change history

  • 05 November 2018

    An outdated version of the Supplementary Information was originally published with this article. In the correct version, the sizing and layout of the figures were adjusted, and the language in the legends was updated for style such that the supplementary legends and the main-text figure legends were stylistically consistent. However, no data were changed or added. The correct version is now available online.


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The study was generously supported in parts by the Deutsche Forschungsgemeinschaft (German Research Foundation, SFB-TR36-project A3 grant to H.-D.V., P.R., M.S.H.), the German Federal Ministry of Education and Research (BCRT grant, all authors), a kick-box grant for young scientists by the Einstein Center for Regenerative Therapies (D.L.W.), and the Berlin Institute of Health medical doctoral research stipend (D.L.W.). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. We would like to acknowledge the assistance of the BCRT Flow & Mass Cytometry Lab, D. Kunkel and J. Hartwig; G. Grütz and the team of the Core Unit Biomarker—Immunological Study Lab, BCRT, Charité—Universitätsmedizin Berlin; M. Streitz for flow cytometry assistance and CMV lysates; K. Vogt for technical assistance with the TSDR analyses; A. Jurisch, K. Grzeschik, and R. Noster for technical assistance; and A. Floriane Hennig and U. Kornak (Institute for Medical Genetics and Human Genetics, Charité – Universitätsmedizin Berlin) for the PX458_T2mali DNA plasmid. We thank the groups of M. Seifert and A. Thiel (both BCRT, Charité—Universitätsmedizin Berlin) for supplying the reagents, and T. Roch and M. Frentsch for critical discussions.

Author information

Author notes

  1. These authors jointly directed this study: Hans-Dieter Volk, Michael Schmueck-Henneresse.


  1. Institute for Medical Immunology, Charité – Universitätsmedizin Berlin, Berlin, Germany

    • Dimitrios L. Wagner
    • , Leila Amini
    • , Desiree J. Wendering
    • , Lisa-Marie Burkhardt
    • , Levent Akyüz
    • , Hans-Dieter Volk
    •  & Michael Schmueck-Henneresse
  2. Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Charité – Universitätsmedizin Berlin, Berlin, Germany

    • Dimitrios L. Wagner
    • , Leila Amini
    • , Desiree J. Wendering
    • , Petra Reinke
    • , Hans-Dieter Volk
    •  & Michael Schmueck-Henneresse
  3. Berlin Institute of Health (BIH), Berlin, Germany

    • Dimitrios L. Wagner
  4. Berlin Center for Advanced Therapies (BeCAT), Charité – Universitätsmedizin Berlin, Berlin, Germany

    • Petra Reinke
    • , Hans-Dieter Volk
    •  & Michael Schmueck-Henneresse


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D.L.W. led the project, designed the research, performed the experiments, analyzed and interpreted the data, and wrote the manuscript. L. Amini and D.J.W. established the methods, performed the experiments, analyzed the data, and revised the manuscript. L.-M.B. performed the experiments. L. Akyüz designed the experimental approaches, and performed the Meso Scale Diagnostics and Ella systems measurements. P.R. wrote the manuscript and supplied the reagents. H.-D.V. designed the research, interpreted the data, and wrote the manuscript. M.S.-H. led the project, designed the research, analyzed and interpreted the data, and wrote the manuscript.

Competing interests

D.L.W., L. Amini, D.J.W., M.S.-H., P.R., and H.-D.V. have a patent pending on CRISPR Associated Protein Reactive T Cell Immunity (European patent application EP18163491.6, 2018). L.-M.B. and L. Akyüz have no financial competing interests.

Corresponding author

Correspondence to Michael Schmueck-Henneresse.

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