A bacteriophage encodes its own CRISPR/Cas adaptive response to evade host innate immunity

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Bacteriophages (or phages) are the most abundant biological entities on earth, and are estimated to outnumber their bacterial prey by tenfold1. The constant threat of phage predation has led to the evolution of a broad range of bacterial immunity mechanisms that in turn result in the evolution of diverse phage immune evasion strategies, leading to a dynamic co-evolutionary arms race2, 3. Although bacterial innate immune mechanisms against phage abound, the only documented bacterial adaptive immune system is the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system, which provides sequence-specific protection from invading nucleic acids, including phage4, 5, 6, 7, 8, 9, 10, 11. Here we show a remarkable turn of events, in which a phage-encoded CRISPR/Cas system is used to counteract a phage inhibitory chromosomal island of the bacterial host. A successful lytic infection by the phage is dependent on sequence identity between CRISPR spacers and the target chromosomal island. In the absence of such targeting, the phage-encoded CRISPR/Cas system can acquire new spacers to evolve rapidly and ensure effective targeting of the chromosomal island to restore phage replication.

At a glance


  1. Genomic organization of the ICP1 CRISPR/Cas system.
    Figure 1: Genomic organization of the ICP1 CRISPR/Cas system.

    a, The ICP1 phage CRISPR/Cas system consists of six cas genes and two CRISPR loci (CR1 and CR2). b, For each CRISPR locus, the repeat (28bp) and spacer (32bp) content is detailed as grey diamonds and coloured rectangles, respectively. Repeats (28bp) that match the repeat consensus are shown in grey diamonds, and degenerate repeats are indicated in hatched grey diamonds. An AT-rich leader sequence precedes each CRISPR locus (grey rectangle). Spacers are coloured according to the percentage identity (solid represent 100% identity, gradient represents 81–97% identity). A fifth ICP1-related phage (ICP1_2003_A) has a genetically identical CRISPR/Cas system to ICP1_2004_A, and has been omitted for simplicity. c, The RNA sequence of the CR1 and CR2 consensus repeat with the partially palindromic sequence forming the predicted stem in the crRNA underlined.

  2. Genomic organization of PLE1, a representative V. cholerae PLE targeted by the CRISPR/Cas system of ICP1-related phages.
    Figure 2: Genomic organization of PLE1, a representative V. cholerae PLE targeted by the CRISPR/Cas system of ICP1-related phages.

    The integrase (int) is in blue, genes encoding hypothetical proteins (with numerical ORF designations) are grey. The locations of protospacers incorporated into the CRISPR locus as spacers 8 and 9 (S8 and S9 of ICP1_2011_A) are indicated in green above the map. The locations of experimentally acquired protospacers are shown below the map in red.

  3. Sequence-based targeting by the ICP1 CRISPR/Cas system is essential for lytic growth on V. cholerae PLE+.
    Figure 3: Sequence-based targeting by the ICP1 CRISPR/Cas system is essential for lytic growth on V. cholerae PLE+.

    a, Disruption of the V. cholerae PLE target protospacer generating V. cholerae PLE(8*). The 32bp protospacer sequence is shaded in grey. b, The sensitivity of each strain (top row) to ICP1 or ICP1(ΔS9) (left column) is shown. Identity between the spacer and targeted protospacer is indicated by the red and blue rectangles. The efficiency of plaquing (EOP, which is the plaque count on the mutant host strain divided by that on the wild-type host strain) is indicated. A dagger indicates that the EOP is 10−5 or 10−8 depending on the presence of PLE in the host strain used for propagation as discussed in the text.

Accession codes

Primary accessions


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Author information


  1. Howard Hughes Medical Institute and Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA

    • Kimberley D. Seed,
    • David W. Lazinski &
    • Andrew Camilli
  2. Division of Infectious Diseases, Massachusetts General Hospital, Boston, Massachusetts 02114, USA

    • Stephen B. Calderwood
  3. Harvard Medical School, Boston, Massachusetts 02114, USA

    • Stephen B. Calderwood


K.D.S. and D.W.L. performed experiments. K.D.S., D.W.L. and A.C. designed experiments. K.D.S. and A.C. wrote the manuscript. S.B.C. provided materials. All authors discussed the results and commented on the manuscript.

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The authors declare no competing financial interests.

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Correspondence to:

The sequences for the CRISPR/Cas system in ICP1_2011_A and ICP1_2006_E have been deposited at GenBank (accession numbers KC152959 and KC152958, respectively). The sequences for the V. cholerae PLEs identified in clinical isolates from the ICDDR,B have been deposited at GenBank/EMBL/DDBJ under the accession numbers KC152960 (PLE1) and KC152961 (PLE2).

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