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Multiplex gene editing by CRISPR–Cpf1 using a single crRNA array

Nature Biotechnology volume 35, pages 31–34 (2017)Cite this article

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An Erratum to this article was published on 08 February 2017

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Abstract

Targeting of multiple genomic loci with Cas9 is limited by the need for multiple or large expression constructs. Here we show that the ability of Cpf1 to process its own CRISPR RNA (crRNA) can be used to simplify multiplexed genome editing. Using a single customized CRISPR array, we edit up to four genes in mammalian cells and three in the mouse brain, simultaneously.

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Figure 1: Cpf1-mediated processing of pre-crRNA is independent of DNA cleavage.
Figure 2: Cpf1-mediated multiplex gene editing in mammalian cells and mouse brain.

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Change history

  • 12 January 2017

    In the version of this article initially published, in Fig. 2j, the percentage for the targets Mecp2, Nlgn3, and Drd1 should be 15.2%, not 16.9%; the same error appeared in the main text, next to last paragraph, “Our results show that ~17%...” should be “Our results show that ~15%....” In the Fig. 2 legend, KASH should be spelled out as “KASH, Klarsicht ANC1 Syne1 homology...” (not “KASH ANC1, Syne homology...”). The errors have been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We would like to thank F.A. Ran for helpful discussions and overall support, and B. Cartigny and J. van den Bogaerde for technical assistance, and the entire Zhang laboratory for support and advice. 6-His-MBP-TEV, a pET-based vector, was kindly given to us by Doug Daniels of the Broad Institute. M.H. was supported by the Human Frontiers Scientific Program. O.A.A. is supported by a Paul and Daisy Soros Fellowship and a Friends of the McGovern Institute Fellowship. J.S.G. is supported by a D.O.E. Computational Science Graduate Fellowship. E.M.D.G. is supported by the National Institute of Biomedical Imaging and Bioengineering (NIBIB), of the National Institutes of Health (5T32EB1680). K.S. is supported by an NIH grant GM10407, Russian Science Foundation grant 14-14-00988, and Skoltech. J.v.d.O. is supported by Netherlands Organization for Scientific Research (NWO) through a TOP grant (714.015.001). F.Z. is supported by the NIH through NIMH (5DP1-MH100706 and 1R01-MH110049); by the New York Stem Cell, Poitras, Simons, Paul G. Allen Family, and Vallee Foundations; and by David R. Cheng, Tom Harriman, and B. Metcalfe. F.Z. is a New York Stem Cell Foundation Robertson Investigator. The authors plan to make the reagents widely available to the academic community through Addgene and to provide software tools via the Zhang lab website (http://www.genome-engineering.org/).

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Author notes

  1. Bernd Zetsche, Matthias Heidenreich and Prarthana Mohanraju: These authors contributed equally to this work.

Authors and Affiliations

  1. Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA

    Bernd Zetsche, Matthias Heidenreich, Iana Fedorova, Jeroen Kneppers, Ellen M DeGennaro, Nerges Winblad, Sourav R Choudhury, Omar O Abudayyeh, Jonathan S Gootenberg, David A Scott & Feng Zhang

  2. McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Bernd Zetsche, Matthias Heidenreich, Iana Fedorova, Nerges Winblad, Sourav R Choudhury, Omar O Abudayyeh, Jonathan S Gootenberg, David A Scott & Feng Zhang

  3. Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Bernd Zetsche, Matthias Heidenreich, Iana Fedorova, Nerges Winblad, Sourav R Choudhury, Omar O Abudayyeh, Jonathan S Gootenberg & Feng Zhang

  4. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Bernd Zetsche, Matthias Heidenreich, Iana Fedorova, Nerges Winblad, Sourav R Choudhury, Omar O Abudayyeh, Jonathan S Gootenberg & Feng Zhang

  5. Department of Developmental Pathology, Institute of Pathology, Bonn Medical School, Bonn, Germany

    Bernd Zetsche

  6. Department of Agrotechnology and Food Sciences, Laboratory of Microbiology, Wageningen University, Wageningen, Netherlands

    Prarthana Mohanraju, Jeroen Kneppers, Wen Y Wu & John van der Oost

  7. Skolkovo Institute of Science and Technology, Skolkovo, Russia

    Iana Fedorova & Konstantin Severinov

  8. Peter the Great St. Petersburg Polytechnic University, St. Petersburg, Russia

    Iana Fedorova

  9. Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Ellen M DeGennaro & Omar O Abudayyeh

  10. Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA

    Jonathan S Gootenberg

  11. Waksman Institute for Microbiology, Rutgers, The State University of New Jersey, Piscataway, New Jersey, USA

    Konstantin Severinov

  12. Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, Russia

    Konstantin Severinov

Authors
  1. Bernd Zetsche
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  2. Matthias Heidenreich
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  3. Prarthana Mohanraju
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  4. Iana Fedorova
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  6. Ellen M DeGennaro
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  9. Omar O Abudayyeh
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  10. Jonathan S Gootenberg
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  11. Wen Y Wu
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  12. David A Scott
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  13. Konstantin Severinov
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  14. John van der Oost
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  15. Feng Zhang
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Contributions

B.Z., M.H., J.v.d.O., and F.Z. conceived this study and designed the experiments. B.Z., M.H., P.M., I.F., J.K., E.M.D., N.W., S.R.C., O.O.A., J.S.G., W.Y.W. and D.A.S. conducted the experiments. K.S., J.v.d.O., and F.Z. supervised this project. B.Z., M.H., J.v.d.O., and F.Z. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to John van der Oost or Feng Zhang.

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Competing interests

A patent has been filed relating to the presented data. F.Z. is a founder and scientific advisor for Editas Medicine, and a scientific advisor for Horizon Discovery.

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Supplementary Figures 1–6 and Supplementary Tables 1–5 (PDF 3028 kb)

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Zetsche, B., Heidenreich, M., Mohanraju, P. et al. Multiplex gene editing by CRISPR–Cpf1 using a single crRNA array. Nat Biotechnol 35, 31–34 (2017). https://doi.org/10.1038/nbt.3737

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