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The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA

Nature volume 468, pages 67–71 (2010)Cite this article

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Abstract

Bacteria and Archaea have developed several defence strategies against foreign nucleic acids such as viral genomes and plasmids. Among them, clustered regularly interspaced short palindromic repeats (CRISPR) loci together with cas (CRISPR-associated) genes form the CRISPR/Cas immune system, which involves partially palindromic repeats separated by short stretches of DNA called spacers, acquired from extrachromosomal elements. It was recently demonstrated that these variable loci can incorporate spacers from infecting bacteriophages and then provide immunity against subsequent bacteriophage infections in a sequence-specific manner. Here we show that the Streptococcus thermophilus CRISPR1/Cas system can also naturally acquire spacers from a self-replicating plasmid containing an antibiotic-resistance gene, leading to plasmid loss. Acquired spacers that match antibiotic-resistance genes provide a novel means to naturally select bacteria that cannot uptake and disseminate such genes. We also provide in vivo evidence that the CRISPR1/Cas system specifically cleaves plasmid and bacteriophage double-stranded DNA within the proto-spacer, at specific sites. Our data show that the CRISPR/Cas immune system is remarkably adapted to cleave invading DNA rapidly and has the potential for exploitation to generate safer microbial strains.

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Figure 1: pNT1 proto-spacers.
Figure 2: The CRISPR1/Cas system in S. thermophilus targets incoming plasmid DNA.
Figure 3: The CRISPR1/Cas system targets bacteriophage DNA.

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Acknowledgements

We thank T. R. Klaenhammer for pTRK687, B. Conway for editorial assistance, D. Tremblay, A. Fleury and B. Stahl for technical assistance. M.-E.D. is the recipient of a Novalait/FQRNT graduate scholarship and A.H.M. of a Clarín/FICYT postdoctoral scholarship. S.M. acknowledges funding from NSERC (Discovery program) and CIHR (Team Grant–Emerging: Novel Alternatives to Antibiotics).

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Authors and Affiliations

  1. Département de biochimie, de microbiologie et de bio-informatique, Faculté des sciences et de génie, Groupe de recherche en écologie buccale, Faculté de médecine dentaire, Félix d’Hérelle Reference Center for Bacterial Viruses, Université Laval, Quebec City, G1V 0A6, Quebec, Canada

    Josiane E. Garneau, Marie-Ève Dupuis, Manuela Villion, Alfonso H. Magadán & Sylvain Moineau

  2. Danisco USA Inc., 3329 Agriculture Drive, Madison, 53716, Wisconsin, USA

    Dennis A. Romero & Rodolphe Barrangou

  3. Danisco France SAS, Boîte Postale 10, F-86220 Dangé-Saint-Romain, France,

    Patrick Boyaval, Christophe Fremaux & Philippe Horvath

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  1. Josiane E. Garneau
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Contributions

S.M. conceived and headed the project. All the authors participated in the design of the study. J.E.G., M.-E.D., M.V. and A.H.M. performed the experiments. J.E.G. and S.M. wrote the main parts of the manuscript. All of the authors analysed the results and commented on the manuscript.

Corresponding author

Correspondence to Sylvain Moineau.

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Competing interests

[Competing interests: D.A.R., R.B., P.B., C.F. and P.H. have submitted patent applications relating to various uses of CRISPR.]

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Garneau, J., Dupuis, MÈ., Villion, M. et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468, 67–71 (2010). https://doi.org/10.1038/nature09523

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