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

Journal name:
Nature
Volume:
468,
Pages:
67–71
Date published:
DOI:
doi:10.1038/nature09523
Received
Accepted
Published online

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.

At a glance

Figures

  1. pNT1 proto-spacers.
    Figure 1: pNT1 proto-spacers.

    The numbers outside and inside the map correspond to proto-spacers from the positive and negative strand, respectively.

  2. The CRISPR1/Cas system in S. thermophilus targets incoming plasmid DNA.
    Figure 2: The CRISPR1/Cas system in S. thermophilus targets incoming plasmid DNA.

    a, Plasmid pNT1 in S. thermophilus strains. Twenty nanograms of plasmid DNA were loaded per lane. pNT1 plasmid was extracted from wild-type strain DGCC7710 (lane 2), from plasmid-interfering mutant (PIM) strain S46 (lane 3), PIM S46::pcas5 (lane 4) and PIM S46::pcas7 (lane 5). pNT1 from lane 2 was linearized with EcoRV (lane 6). Lanes 1 and 7: supercoiled and 1-kb DNA ladders (Invitrogen), respectively. b, Southern hybridization of plasmid pNT1 in S. thermophilus strains. Lane 1: 1ng of native pNT1 plasmid extracted from wild-type strain DGCC7710. Lane 2: 10ng of pNT1 extracted from PIM S46. The DIG-labelled probe 6 (Supplementary Table 2) was used. c, Direct sequencing electropherograms from primers NT17215_3 (upper part) and NT17225_2 (lower part) (see Methods). The non-templated addition of adenine (T in the reverse complement sequence shown here) at the extremity of the primer NT17225_2 sequence is a sequencing artefact due to the polymerase35.

  3. The CRISPR1/Cas system targets bacteriophage DNA.
    Figure 3: The CRISPR1/Cas system targets bacteriophage DNA.

    a, Bacteriophage 2972 genome and position of proto-spacers. Light grey, dark grey and black arrows indicate early, middle and late transcription module, respectively28. Proto-spacers above and below the genome indicate positive and negative strands, respectively. b, Restriction fragments detected by hybridization and position of probes (1, 2 and 3). c, Southern blots of bacteriophage-infected strains DGCC7710, BIM S4, BIM S7, BIM S4/S32, BIM S4::pcas5 and BIM S4::pcas7 over 45min (conducted at 2min, 15min, 30min and 45min). NI, non-infected strain. C+, positive control, 10ng of digested DNA from bacteriophage 2972. One microgram of total DNA from DGCC7710 and 10μg of bacteriophage-insensitive mutant per lane.

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Author information

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, Quebec G1V 0A6, Canada

    • Josiane E. Garneau,
    • Marie-Ève Dupuis,
    • Manuela Villion,
    • Alfonso H. Magadán &
    • Sylvain Moineau
  2. Danisco USA Inc., 3329 Agriculture Drive, Madison, Wisconsin 53716, 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

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.

Competing financial 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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Supplementary information

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  1. Supplementary Information (506K)

    This file contains Supplementary Figures 1-2 with legends and Supplementary Tables 1-3.

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