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Abstract

The RNA-guided endonuclease Cpf1 is a promising tool for genome editing in eukaryotic cells1,2,3,4,5,6,7. However, the utility of the commonly used Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1) and Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1) is limited by their requirement of a TTTV protospacer adjacent motif (PAM) in the DNA substrate. To address this limitation, we performed a structure-guided mutagenesis screen to increase the targeting range of Cpf1. We engineered two AsCpf1 variants carrying the mutations S542R/K607R and S542R/K548V/N552R, which recognize TYCV and TATV PAMs, respectively, with enhanced activities in vitro and in human cells. Genome-wide assessment of off-target activity using BLISS7 indicated that these variants retain high DNA-targeting specificity, which we further improved by introducing an additional non-PAM-interacting mutation. Introducing the identified PAM-interacting mutations at their corresponding positions in LbCpf1 similarly altered its PAM specificity. Together, these variants increase the targeting range of Cpf1 by approximately threefold in human coding sequences to one cleavage site per 11 bp.

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Change history

  • 08 June 2017

    In the version of this article initially published, the accession code given—SRR5611789—was for one sample only, rather than for the entire study. The study code is SRP108089. The error has been corrected for the print, PDF and HTML versions of this article.

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Acknowledgements

We thank A. Magnell for experimental assistance; R. Macrae for a critical review of the manuscript; and the entire Zhang laboratory for support and advice. D.B.T.C. is supported by T32GM007753 from the National Institute of General Medical Sciences. W.X.Y. is supported by T32GM007753 from the National Institute of General Medical Sciences and a Paul and Daisy Soros Fellowship. J.C.M. is supported by the NIH (training grant 2 T32 GM 7287-41). H.N. is supported by JST, PRESTO (JPMJPR13L8), JSPS KAKENHI (Grant Numbers 26291010 and 15H01463). O.N. is supported by the Basic Science and Platform Technology Program for Innovative Biological Medicine from the Japan Agency for Medical Research and Development, AMED, and the Council for Science, and Platform for Drug Discovery, Informatics, and Structural Life Science from the Ministry of Education, Culture, Sports, Science and Technology. N.C. is supported by the Karolinska Institutet, the Swedish Research Council (521-2014-2866), the Swedish Cancer Research Foundation (CAN 2015/585), and the Ragnar Söderberg Foundation. F.Z. is a New York Stem Cell Foundation–Robertson Investigator. F.Z. is supported by the NIH through NIMH (5DP1-MH100706 and 1R01-MH110049), NSF, Howard Hughes Medical Institute, the New York Stem Cell, Simons, Paul G. Allen Family, and Vallee Foundations; and James and Patricia Poitras, Robert Metcalfe, and David Cheng.

Author information

Affiliations

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

    • Linyi Gao
    • , David B T Cox
    • , Winston X Yan
    • , Martin W Schneider
    •  & Feng Zhang
  2. Department of Biological Engineering, Massachusetts Institute of Technology Cambridge, Massachusetts, USA.

    • Linyi Gao
    •  & Feng Zhang
  3. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • David B T Cox
    •  & John C Manteiga
  4. Harvard-MIT Division of Health Sciences and Technology, Harvard Medical School, Boston, Massachusetts, USA.

    • David B T Cox
    •  & Winston X Yan
  5. Graduate Program in Biophysics, Harvard Medical School, Boston, Massachusetts, USA.

    • Winston X Yan
  6. Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan.

    • Takashi Yamano
    • , Hiroshi Nishimasu
    •  & Osamu Nureki
  7. JST, PRESTO, Tokyo, Japan.

    • Hiroshi Nishimasu
  8. Science for Life Laboratory, Division of Translational Medicine and Chemical Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.

    • Nicola Crosetto
  9. McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Feng Zhang
  10. Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Feng Zhang

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Contributions

L.G., D.B.T.C., and F.Z. conceived this study. L.G. and D.B.T.C. performed experiments with help from all authors. J.C.M. contributed to the bacterial selection screen. M.W.S. processed BLISS samples, and W.X.Y. analyzed BLISS data. T.Y., H.N., and O.N. provided unpublished AsCpf1 crystal structure information. N.C. provided an unpublished BLISS protocol. F.Z. supervised research. L.G. and F.Z. wrote the manuscript with input from all authors.

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. L.G., D.B.T.C., and F.Z. are co-inventors on US Provisional Patent Application Serial No. 62/324,820, directed to the Cpf1 protein variants, as described in this manuscript.

Corresponding author

Correspondence to Feng Zhang.

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DOI

https://doi.org/10.1038/nbt.3900

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