Letter | Published:

New CRISPR–Cas systems from uncultivated microbes

Nature volume 542, pages 237241 (09 February 2017) | Download Citation

Abstract

CRISPR–Cas systems provide microbes with adaptive immunity by employing short DNA sequences, termed spacers, that guide Cas proteins to cleave foreign DNA1,2. Class 2 CRISPR–Cas systems are streamlined versions, in which a single RNA-bound Cas protein recognizes and cleaves target sequences3,4. The programmable nature of these minimal systems has enabled researchers to repurpose them into a versatile technology that is broadly revolutionizing biological and clinical research5. However, current CRISPR–Cas technologies are based solely on systems from isolated bacteria, leaving the vast majority of enzymes from organisms that have not been cultured untapped. Metagenomics, the sequencing of DNA extracted directly from natural microbial communities, provides access to the genetic material of a huge array of uncultivated organisms6,7. Here, using genome-resolved metagenomics, we identify a number of CRISPR–Cas systems, including the first reported Cas9 in the archaeal domain of life, to our knowledge. This divergent Cas9 protein was found in little-studied nanoarchaea as part of an active CRISPR–Cas system. In bacteria, we discovered two previously unknown systems, CRISPR–CasX and CRISPR–CasY, which are among the most compact systems yet discovered. Notably, all required functional components were identified by metagenomics, enabling validation of robust in vivo RNA-guided DNA interference activity in Escherichia coli. Interrogation of environmental microbial communities combined with in vivo experiments allows us to access an unprecedented diversity of genomes, the content of which will expand the repertoire of microbe-based biotechnologies.

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Acknowledgements

We thank N. Ma, K. Zhou and D. McGrath for technical assistance; C. Brown, M. Olm, M. O’Connell, J. Chen and S. Floor for reading the manuscript and discussions; and V. Yu for the S. cerevisiae expression strain. D.B. was supported by a long-term EMBO fellowship, L.B.H. by a US National Science Foundation Graduate Research Fellowship, and A.J.P. by a fellowship of the German Science Foundation (DFG PR 1603/1-1). J.A.D. is an Investigator of the Howard Hughes Medical Institute. This research was supported in part by the Allen Distinguished Investigator Program, through The Paul G. Allen Frontiers Group, the National Science Foundation (MCB-1244557 to J.A.D.) and the Lawrence Berkeley National Laboratory’s Sustainable Systems Scientific Focus Area funded by the US Department of Energy (DE-AC02-05CH11231 to J.F.B.). DNA sequencing was conducted at the DOE Joint Genome Institute, a DOE Office of Science User Facility, via the Community Science Program.

Author information

Author notes

    • David Burstein
    • , Lucas B. Harrington
    •  & Steven C. Strutt

    These authors contributed equally to this work.

Affiliations

  1. Department of Earth and Planetary Sciences, University of California, Berkeley, California 94720, USA

    • David Burstein
    • , Alexander J. Probst
    • , Karthik Anantharaman
    • , Brian C. Thomas
    •  & Jillian F. Banfield
  2. Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA

    • Lucas B. Harrington
    • , Steven C. Strutt
    •  & Jennifer A. Doudna
  3. Department of Chemistry, University of California, Berkeley, California 94720, USA

    • Jennifer A. Doudna
  4. Howard Hughes Medical Institute, University of California, Berkeley, California 94720, USA

    • Jennifer A. Doudna
  5. Innovative Genomics Initiative, University of California, Berkeley, California 94720, USA

    • Jennifer A. Doudna
  6. MBIB Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA

    • Jennifer A. Doudna
  7. Department of Environmental Science, Policy, and Management, University of California, Berkeley, California 94720, USA

    • Jillian F. Banfield

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Contributions

D.B., L.B.H., S.C.S., J.A.D. and J.F.B. designed the study and wrote the manuscript. A.J.P., K.A., J.F.B., B.T.C. and D.B. assembled the data and reconstructed the genomes. D.B., L.B.H., S.C.S. and J.F.B. computationally analysed the CRISPR–Cas systems. L.B.H. and D.B. designed and executed experimental work with CRISPR–CasX and CRISPR–CasY. S.C.S. designed and executed the experimental work with ARMAN Cas9. The manuscript was read, edited and approved by all authors.

Competing interests

The Regents of the University of California have filed a provisional patent application related to the technology described in this work to the United States Patent and Trademark Office, in which D.B., L.B.H., S.C.S., J.A.D. and J.F.B. are listed as inventors.

Corresponding authors

Correspondence to Jennifer A. Doudna or Jillian F. Banfield.

Reviewer Information Nature thanks E. Sontheimer, R. Sorek and M. White for their contribution to the peer review of this work.

Extended data

Supplementary information

Excel files

  1. 1.

    Supplementary Table 1

    This file contains Supplementary Table 1, reconstructed spacer and protospacers of the ARMAN-1 Type II CRISPR-Cas system.

  2. 2.

    Supplementary Table 2

    This file contains Supplementary Table 2, a list of primers and plasmids used in the study.

Zip files

  1. 1.

    Supplementary Data

    This zipped file contains Supplementary Data sets 1-6.

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DOI

https://doi.org/10.1038/nature21059

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