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

An Erratum to this article was published on 08 February 2017

This article has been updated

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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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.

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Correspondence to John van der Oost or Feng Zhang.

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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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