Letter | Published:

Engineered CRISPR-Cas9 nucleases with altered PAM specificities

Nature volume 523, pages 481485 (23 July 2015) | Download Citation

Abstract

Although CRISPR-Cas9 nucleases are widely used for genome editing1,2, the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM)3,4,5,6. As a result, it can often be difficult to target double-stranded breaks (DSBs) with the precision that is necessary for various genome-editing applications. The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation. Here we show that the commonly used Streptococcus pyogenes Cas9 (SpCas9) can be modified to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design. These altered PAM specificity variants enable robust editing of endogenous gene sites in zebrafish and human cells not currently targetable by wild-type SpCas9, and their genome-wide specificities are comparable to wild-type SpCas9 as judged by GUIDE-seq analysis7. In addition, we identify and characterize another SpCas9 variant that exhibits improved specificity in human cells, possessing better discrimination against off-target sites with non-canonical NAG and NGA PAMs and/or mismatched spacers. We also find that two smaller-size Cas9 orthologues, Streptococcus thermophilus Cas9 (St1Cas9) and Staphylococcus aureus Cas9 (SaCas9), function efficiently in the bacterial selection systems and in human cells, suggesting that our engineering strategies could be extended to Cas9s from other species. Our findings provide broadly useful SpCas9 variants and, more importantly, establish the feasibility of engineering a wide range of Cas9s with altered and improved PAM specificities.

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Accessions

Primary accessions

Sequence Read Archive

Data deposits

All new reagents described in this work have been deposited with the non-profit plasmid distribution service Addgene (http://www.addgene.org/crispr-cas). A web-tool to design sgRNA sites for the engineered variants and orthogonal Cas9 nucleases described in this study can be found at http://www.CasBLASTR.org. The sequences generated in this study have been deposited in the Sequences Read Archive under accession number SRP058629.

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Acknowledgements

We thank D. Edgell for providing the bacterial strain and plasmids related to the bacterial selection; J. Angstman and V. Pattanayak for discussion and comments on the manuscript. 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., NIH R01 GM088040 to J.K.J. and R.T.P., The Jim and Ann Orr Research Scholar Award (to J.K.J.), and a National Sciences and Engineering Research Council of Canada Postdoctoral Fellowship (to B.P.K.).

Author information

Affiliations

  1. Molecular Pathology Unit & Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA

    • Benjamin P. Kleinstiver
    • , Michelle S. Prew
    • , Shengdar Q. Tsai
    • , Ved V. Topkar
    • , Nhu T. Nguyen
    • , Zongli Zheng
    • , Martin J. Aryee
    •  & J. Keith Joung
  2. Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA

    • Benjamin P. Kleinstiver
    • , Michelle S. Prew
    • , Shengdar Q. Tsai
    • , Ved V. Topkar
    • , Nhu T. Nguyen
    •  & J. Keith Joung
  3. Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Benjamin P. Kleinstiver
    • , Shengdar Q. Tsai
    • , Zongli Zheng
    • , Martin J. Aryee
    •  & J. Keith Joung
  4. Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm SE-171 77, Sweden

    • Zongli Zheng
  5. Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA

    • Andrew P. W. Gonzales
    • , Zhuyun Li
    • , Randall T. Peterson
    •  & Jing-Ruey Joanna Yeh
  6. Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Andrew P. W. Gonzales
    •  & Randall T. Peterson
  7. Broad Institute, Cambridge, Massachusetts 02142, USA

    • Andrew P. W. Gonzales
    •  & Randall T. Peterson
  8. Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Jing-Ruey Joanna Yeh
  9. Department of Biostatistics, Harvard T.H. Chan School of Public Health, Boston, Massachusetts 02115, USA

    • Martin J. Aryee

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Contributions

B.P.K., M.S.P., S.Q.T. and N.T.N. performed all bacterial and human cell-based experiments. A.P.W.G. and Z.L. performed all zebrafish experiments. S.Q.T., V.T., Z.Z. and M.J.A. analysed the site-depletion, targeted deep-sequencing, and GUIDE-seq data. B.P.K., R.T.P., J.-R.J.Y. and J.K.J. directed the research and interpreted experiments. B.P.K. and J.K.J. wrote the manuscript with input from all the authors.

Competing interests

J.K.J. is a consultant for Horizon Discovery. J.K.J. has financial interests in Editas Medicine, Hera Testing Laboratories, Poseida Therapeutics, and Transposagen Biopharmaceuticals. J.K.J.’s interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies.

Corresponding author

Correspondence to J. Keith Joung.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains Supplementary Sequences and details of the Supplementary software for analyzing PAM depletion MiSeq data.

Excel files

  1. 1.

    Supplementary Table 1

    This table shows the Oligos used to generate positive and negative selection plasmids.

  2. 2.

    Supplementary Table 2

    This file contains S. pyogenes sgRNAs targets.

  3. 3.

    Supplementary Table 3

    This file contains the PAM MiSeq data.

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DOI

https://doi.org/10.1038/nature14592

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