Letter | Published:

Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells

Nature Biotechnology volume 34, pages 869874 (2016) | Download Citation

Abstract

The activities and genome-wide specificities of CRISPR-Cas Cpf1 nucleases1 are not well defined. We show that two Cpf1 nucleases from Acidaminococcus sp. BV3L6 and Lachnospiraceae bacterium ND2006 (AsCpf1 and LbCpf1, respectively) have on-target efficiencies in human cells comparable with those of the widely used Streptococcus pyogenes Cas9 (SpCas9)2,3,4,5. We also report that four to six bases at the 3′ end of the short CRISPR RNA (crRNA) used to program Cpf1 nucleases are insensitive to single base mismatches, but that many of the other bases in this region of the crRNA are highly sensitive to single or double substitutions. Using GUIDE-seq and targeted deep sequencing analyses performed with both Cpf1 nucleases, we were unable to detect off-target cleavage for more than half of 20 different crRNAs. Our results suggest that AsCpf1 and LbCpf1 are highly specific in human cells.

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Acknowledgements

This work was supported by a National Institutes of Health (NIH) Director's Pioneer Award (DP1 GM105378) and NIH R01 GM107427 to J.K.J., the Jim and Ann Orr Research Scholar Award (to J.K.J.), a Natural Sciences and Engineering Research Council of Canada Postdoctoral Fellowship (to B.P.K.), an award from the Massachusetts General Hospital Collaborative Center for X-Linked Dystonia-Parkinsonism, and an MGH Tosteson Award (to S.Q.T.). New reagents described in this work have been deposited with the non-profit plasmid distribution service Addgene (http://www.addgene.org/crispr-cas).

Author information

Author notes

    • Benjamin P Kleinstiver
    •  & Shengdar Q Tsai

    These authors contributed equally to this work.

Affiliations

  1. Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, Massachusetts, USA.

    • Benjamin P Kleinstiver
    • , Shengdar Q Tsai
    • , Michelle S Prew
    • , Nhu T Nguyen
    • , Moira M Welch
    • , Jose M Lopez
    • , Zachary R McCaw
    • , Martin J Aryee
    •  & J Keith Joung
  2. Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts, USA.

    • Benjamin P Kleinstiver
    • , Shengdar Q Tsai
    • , Michelle S Prew
    • , Nhu T Nguyen
    • , Moira M Welch
    • , Jose M Lopez
    • , Zachary R McCaw
    • , Martin J Aryee
    •  & J Keith Joung
  3. Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA.

    • Benjamin P Kleinstiver
    • , Shengdar Q Tsai
    • , Michelle S Prew
    • , Nhu T Nguyen
    • , Moira M Welch
    • , Jose M Lopez
    •  & J Keith Joung
  4. Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA.

    • Benjamin P Kleinstiver
    • , Shengdar Q Tsai
    • , Jose M Lopez
    • , Martin J Aryee
    •  & J Keith Joung
  5. Department of Biostatistics, Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USA.

    • Zachary R McCaw
    •  & Martin J Aryee

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Contributions

B.P.K., S.Q.T., and J.K.J. conceived of and designed experiments. B.P.K., S.Q.T., M.S.P., N.T.N., and M.M.W. performed all human cell experiments and analyzed data. S.Q.T., J.M.L., Z.R.M., and M.J.A. analyzed the GUIDE-seq and targeted deep-sequencing data. B.P.K., S.Q.T., and J.K.J. wrote the manuscript with input from all authors.

Competing interests

J.K.J. is a consultant for Horizon Discovery. J.K.J. has financial interests in Beacon Genomics, Editas Medicine, Hera Testing Laboratories, Poseida Therapeutics, and Transposagen Biopharmaceuticals. JKJ's interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies. S.Q.T., M.J.A., and J.K.J. are co-founders of Beacon Genomics, a company that is commercializing methods for determining nuclease specificity.

Corresponding authors

Correspondence to Benjamin P Kleinstiver or J Keith Joung.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–8 and Supplementary Note 1

Excel files

  1. 1.

    Supplementary Table 1

    gRNA target sites.

  2. 2.

    Supplementary Table 2

    In Silico off-targets.

  3. 3.

    Supplementary Table 3

    Deep sequencing data and p-values.

  4. 4.

    Supplementary Table 4

    GUIDE-seq data.

  5. 5.

    Supplementary Table 5

    Oligonucleotides.

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DOI

https://doi.org/10.1038/nbt.3620

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