Article | Published:

Evolved Cas9 variants with broad PAM compatibility and high DNA specificity

Nature volume 556, pages 5763 (05 April 2018) | Download Citation

Abstract

A key limitation of the use of the CRISPR–Cas9 system for genome editing and other applications is the requirement that a protospacer adjacent motif (PAM) be present at the target site. For the most commonly used Cas9 from Streptococcus pyogenes (SpCas9), the required PAM sequence is NGG. No natural or engineered Cas9 variants that have been shown to function efficiently in mammalian cells offer a PAM less restrictive than NGG. Here we use phage-assisted continuous evolution to evolve an expanded PAM SpCas9 variant (xCas9) that can recognize a broad range of PAM sequences including NG, GAA and GAT. The PAM compatibility of xCas9 is the broadest reported, to our knowledge, among Cas9 proteins that are active in mammalian cells, and supports applications in human cells including targeted transcriptional activation, nuclease-mediated gene disruption, and cytidine and adenine base editing. Notably, despite its broadened PAM compatibility, xCas9 has much greater DNA specificity than SpCas9, with substantially lower genome-wide off-target activity at all NGG target sites tested, as well as minimal off-target activity when targeting genomic sites with non-NGG PAMs. These findings expand the DNA targeting scope of CRISPR systems and establish that there is no necessary trade-off between Cas9 editing efficiency, PAM compatibility and DNA specificity.

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Acknowledgements

We thank A. Badran, B. Hubbard, J. Levy, T. Huang, G. Church, A. Chavez, K. Esvelt, S. Vora and J. Scheiman for discussions. This work was supported by DARPA HR0011-17-2-0049, US NIH RM1 HG009490, R01 EB022376 and R35 GM118062 and the HHMI. J.H.H. was supported by NDSEG and NSF graduate fellowships. S.M.M. was supported by an NSF graduate fellowship. W.T. is an HHMI Fellow of the Jane Coffin Childs Memorial Fund. L.C. was supported by the Agency for Science, Technology, and Research, Singapore.

Author information

Affiliations

  1. Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA

    • Johnny H. Hu
    • , Shannon M. Miller
    • , Maarten H. Geurts
    • , Weixin Tang
    • , Liwei Chen
    • , Ning Sun
    • , Christina M. Zeina
    • , Xue Gao
    • , Holly A. Rees
    • , Zhi Lin
    •  & David R. Liu
  2. Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts 02138, USA

    • Johnny H. Hu
    • , Shannon M. Miller
    • , Maarten H. Geurts
    • , Weixin Tang
    • , Liwei Chen
    • , Ning Sun
    • , Christina M. Zeina
    • , Xue Gao
    • , Holly A. Rees
    • , Zhi Lin
    •  & David R. Liu
  3. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA

    • Johnny H. Hu
    • , Shannon M. Miller
    • , Maarten H. Geurts
    • , Weixin Tang
    • , Liwei Chen
    • , Ning Sun
    • , Christina M. Zeina
    • , Xue Gao
    • , Holly A. Rees
    • , Zhi Lin
    •  & David R. Liu

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Contributions

J.H.H. designed the research, performed PACE, characterized variants in bacteria, conducted human cell experiments, analysed data, performed off-target analysis and wrote the manuscript. S.M.M. performed human cell experiments, analysed data and wrote the manuscript. M.H.G. performed human cell experiments and data analysis. W.T. performed human cell experiments and cloning. L.C. and C.M.Z. assisted with cloning and PACE. N.S. optimized Cas9 PACE. X.G. assisted with off-target analysis. H.A.R. assisted with indel and base-editing analyses. Z.L. assisted with human cell experiments. D.R.L. designed and supervised the research and wrote the manuscript.

Competing interests

J.H.H. and D.R.L. have filed patent applications on this work (WO2017070633). D.R.L. is a consultant and co-founder of Editas Medicine, Beam Therapeutics, and Pairwise Plants, companies that use genome editing technologies. The authors declare no other competing interests.

Corresponding author

Correspondence to David R. Liu.

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    Supplementary Information

    This file contains the data for Supplementary Tables 1-17, Supplementary Sequences 1-3 and Supplementary Notes 1-3.

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DOI

https://doi.org/10.1038/nature26155

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