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CRISPR–Cas systems exploit viral DNA injection to establish and maintain adaptive immunity

Nature volume 544, pages 101104 (06 April 2017) | Download Citation


Clustered regularly interspaced short palindromic repeats (CRISPR)–Cas systems provide protection against viral1 and plasmid2 infection by capturing short DNA sequences from these invaders and integrating them into the CRISPR locus of the prokaryotic host1. These sequences, known as spacers, are transcribed into short CRISPR RNA guides3,4,5 that specify the cleavage site of Cas nucleases in the genome of the invader6,7,8. It is not known when spacer sequences are acquired during viral infection. Here, to investigate this, we tracked spacer acquisition in Staphylococcus aureus cells harbouring a type II CRISPR–Cas9 system after infection with the staphylococcal bacteriophage ϕ12. We found that new spacers were acquired immediately after infection preferentially from the cos site, the viral free DNA end that is first injected into the cell. Analysis of spacer acquisition after infection with mutant phages demonstrated that most spacers are acquired during DNA injection, but not during other stages of the viral cycle that produce free DNA ends, such as DNA replication or packaging. Finally, we showed that spacers acquired from early-injected genomic regions, which direct Cas9 cleavage of the viral DNA immediately after infection, provide better immunity than spacers acquired from late-injected regions. Our results reveal that CRISPR–Cas systems exploit the phage life cycle to generate a pattern of spacer acquisition that ensures a successful CRISPR immune response.

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We thank R. Heler for plasmid pRH163; J. McGinn for plasmid pJM62; J. Penades for providing ϕ12 as well as assistance in working with it; B. Dujon for permission to use the I-SceI endonuclease; and The Rockefeller University Genomics Resource Center core facility for performing next-generation sequencing. J.W.M. is a Fellow of The Jane Coffin Childs Memorial Fund for Medical Research. L.A.M. is supported by the Rita Allen Scholars Program, an Irma T. Hirschl Award, a Sinsheimer Foundation Award, a Burroughs Wellcome Fund PATH award, an NIH Director’s New Innovator Award (1DP2AI104556-01) and an HHMI-Simons Faculty Scholar Award.

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  1. Laboratory of Bacteriology, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA

    • Joshua W. Modell
    • , Wenyan Jiang
    •  & Luciano A. Marraffini


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J.W.M. and L.A.M. conceived the study and designed experiments. J.W.M. and W.J. designed the spacer library construction method. W.J. performed the CRISPR immunization simulation assay. All other work was executed by J.W.M. L.A.M. and J.W.M. wrote the paper with the help of W.J.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Luciano A. Marraffini.

Reviewer Information Nature thanks R. Barrangou, J. Doudna and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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