Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Bacteriophage genes that inactivate the CRISPR/Cas bacterial immune system


A widespread system used by bacteria for protection against potentially dangerous foreign DNA molecules consists of the clustered regularly interspaced short palindromic repeats (CRISPR) coupled with cas (CRISPR-associated) genes1. Similar to RNA interference in eukaryotes2, these CRISPR/Cas systems use small RNAs for sequence-specific detection and neutralization of invading genomes3. Here we describe the first examples of genes that mediate the inhibition of a CRISPR/Cas system. Five distinct ‘anti-CRISPR’ genes were found in the genomes of bacteriophages infecting Pseudomonas aeruginosa. Mutation of the anti-CRISPR gene of a phage rendered it unable to infect bacteria with a functional CRISPR/Cas system, and the addition of the same gene to the genome of a CRISPR/Cas-targeted phage allowed it to evade the CRISPR/Cas system. Phage-encoded anti-CRISPR genes may represent a widespread mechanism for phages to overcome the highly prevalent CRISPR/Cas systems. The existence of anti-CRISPR genes presents new avenues for the elucidation of CRISPR/Cas functional mechanisms and provides new insight into the co-evolution of phages and bacteria.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: The CRISPR/Cas system is inhibited by expression of phage genes.
Figure 2: An anti-CRISPR gene protects phages from the CRISPR/Cas system during infection.

Accession codes

Primary accessions

NCBI Reference Sequence

Data deposits

Phage genomes have been deposited in the National Center for Biotechnology Information under accession numbers JX434030 (JBD5), JX434031 (JBD24), JX434032 (JBD30) and JX434033 (JBD88a).


  1. Makarova, K. S. et al. Evolution and classification of the CRISPR-Cas systems. Nature Rev. Microbiol. 9, 467–477 (2011)

    CAS  Article  Google Scholar 

  2. Makarova, K. S., Grishin, N. V., Shabalina, S. A., Wolf, Y. I. & Koonin, E. V. A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biol. Direct 1, 7 (2006)

    Article  Google Scholar 

  3. Brouns, S. J. J. et al. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321, 960–964 (2008)

    ADS  CAS  Article  Google Scholar 

  4. Rodriguez-Valera, F. et al. Explaining microbial population genomics through phage predation. Nature Rev. Microbiol. 7, 828–836 (2009)

    CAS  Article  Google Scholar 

  5. Labrie, S. J., Samson, J. E. & Moineau, S. Bacteriophage resistance mechanisms. Nature Rev. Microbiol. 8, 317–327 (2010)

    CAS  Article  Google Scholar 

  6. Jore, M. M., Brouns, S. J. J. & van der Oost, J. RNA in defense: CRISPRs protect prokaryotes against mobile genetic elements. Cold Spring Harb. Perspect. Biol. 4, (2012)

  7. Haurwitz, R. E., Jinek, M., Wiedenheft, B., Zhou, K. & Doudna, J. A. Sequence- and structure-specific RNA processing by a CRISPR endonuclease. Science 329, 1355–1358 (2010)

    ADS  CAS  Article  Google Scholar 

  8. Deltcheva, E. et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471, 602–607 (2011)

    ADS  CAS  Article  Google Scholar 

  9. Wiedenheft, B. et al. Structures of the RNA-guided surveillance complex from a bacterial immune system. Nature 477, 486–489 (2011)

    ADS  CAS  Article  Google Scholar 

  10. Westra, E. R. et al. CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3. Mol. Cell 46, 595–605 (2012)

    CAS  Article  Google Scholar 

  11. Garneau, J. E. et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468, 67–71 (2010)

    ADS  CAS  Article  Google Scholar 

  12. Barrangou, R. et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 1709–1712 (2007)

    ADS  CAS  Article  Google Scholar 

  13. Yosef, I., Goren, M. G. & Qimron, U. Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli. Nucleic Acids Res. 40, 5569–5576 (2012)

    CAS  Article  Google Scholar 

  14. Cady, K. C., Bondy-Denomy, J., Heussler, G. E., Davidson, A. R. & O’Toole, G. A. The CRISPR/Cas adaptive immune system of Pseudomonas aeruginosa mediates resistance to naturally occurring and engineered phages. J. Bacteriol. 194, 5728–5738 (2012)

    CAS  Article  Google Scholar 

  15. Marraffini, L. A. & Sontheimer, E. J. Self versus non-self discrimination during CRISPR RNA-directed immunity. Nature 463, 568–571 (2010)

    ADS  CAS  Article  Google Scholar 

  16. Mojica, F. J. M., Diez-Villasenor, C., Garcia-Martinez, J. & Almendros, C. Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology 155, 733–740 (2009)

    CAS  Article  Google Scholar 

  17. Braid, M. D., Silhavy, J. L., Kitts, C. L., Cano, R. J. & Howe, M. M. Complete genomic sequence of bacteriophage B3, a Mu-like phage of Pseudomonas aeruginosa. J. Bacteriol. 186, 6560–6574 (2004)

    CAS  Article  Google Scholar 

  18. Morgan, G. J., Hatfull, G. F., Casjens, S. & Hendrix, R. W. Bacteriophage Mu genome sequence: analysis and comparison with Mu-like prophages in Haemophilus, Neisseria and Deinococcus. J. Mol. Biol. 317, 337–359 (2002)

    CAS  Article  Google Scholar 

  19. Datsenko, K. A. et al. Molecular memory of prior infections activates the CRISPR/Cas adaptive bacterial immunity system. Nature Commun. 3, 945 (2012)

    ADS  Article  Google Scholar 

  20. Cady, K. C. & O’Toole, G. A. Non-identity-mediated CRISPR-bacteriophage interaction mediated via the Csy and Cas3 proteins. J. Bacteriol. 193, 3433–3445 (2011)

    CAS  Article  Google Scholar 

  21. Battle, S. E., Meyer, F., Rello, J., Kung, V. L. & Hauser, A. R. Hybrid pathogenicity island PAGI-5 contributes to the highly virulent phenotype of a Pseudomonas aeruginosa isolate in mammals. J. Bacteriol. 190, 7130–7140 (2008)

    CAS  Article  Google Scholar 

  22. Cady, K. C. et al. Prevalence, conservation and functional analysis of Yersinia and Escherichia CRISPR regions in clinical Pseudomonas aeruginosa isolates. Microbiology 157, 430–437 (2011)

    CAS  Article  Google Scholar 

  23. Semenova, E. et al. Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence. Proc. Natl Acad. Sci. USA 108, 10098–10103 (2011)

    ADS  CAS  Article  Google Scholar 

  24. Zegans, M. E. et al. Interaction between bacteriophage DMS3 and host CRISPR region inhibits group behaviors of Pseudomonas aeruginosa. J. Bacteriol. 191, 210–219 (2009)

    CAS  Article  Google Scholar 

  25. Heo, Y.-J., Chung, I.-Y., Choi, K. B., Lau, G. W. & Cho, Y.-H. Genome sequence comparison and superinfection between two related Pseudomonas aeruginosa phages, D3112 and MP22. Microbiology 153, 2885–2895 (2007)

    CAS  Article  Google Scholar 

  26. Chung, I.-Y. & Cho, Y.-H. Complete genome sequences of two Pseudomonas aeruginosa temperate phages, MP29 and MP42, which lack the phage-host CRISPR interaction. J. Virol. 86, 8336 (2012)

    CAS  Article  Google Scholar 

  27. Wang, P. W., Chu, L. & Guttman, D. S. Complete sequence and evolutionary genomic analysis of the Pseudomonas aeruginosa transposable bacteriophage D3112. J. Bacteriol. 186, 400–410 (2004)

    CAS  Article  Google Scholar 

  28. Aziz, R. K. et al. The RAST server: rapid annotations using subsystems technology. BMC Genomics 9, 75 (2008)

    Article  Google Scholar 

  29. Larkin, M. A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007)

    CAS  Article  Google Scholar 

  30. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990)

    CAS  Article  Google Scholar 

  31. Qiu, D., Damron, F. H., Mima, T., Schweizer, H. P. & Yu, H. D. PBAD-based shuttle vectors for functional analysis of toxic and highly regulated genes in Pseudomonas and Burkholderia spp. and other bacteria. Appl. Environ. Microbiol. 74, 7422–7426 (2008)

    CAS  Article  Google Scholar 

Download references


We thank D. Guttman, Y.-H. Cho, K. Cady and G. O’Toole for providing P. aeruginosa strains and phages. We also thank K. Severinov for providing the M13 phage and E. coli strains required for assaying the type 1-E system. We thank J. Brumell, A. Spence and W. Navarre for reading the manuscript. We also thank D. Bona for technical assistance. This work was supported by an Operating Grant to K.L.M. (fund number MOP- 6279) and an Emerging Team Grant to A.R.D. and K.L.M. (fund number XNE86943), both of which were from the Canadian Institutes for Health Research. J.B.D. was supported by a CIHR Canada Graduate Scholarship Doctoral Award.

Author information

Authors and Affiliations



J.B.-D. designed experiments, performed experiments and wrote the manuscript, A.P. performed experiments, K.L.M. supervised experiments, and A.R.D. designed experiments and wrote the manuscript.

Corresponding author

Correspondence to Alan R. Davidson.

Ethics declarations

Competing interests

The authors have filed a provisional patent pertaining to biotechnological applications of anti-CRISPR genes.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-12, Supplementary Tables 1-2 and a Supplementary Reference. (PDF 3310 kb)

Supplementary Data

This file contains Supplementary Table 3 with a, Bacteria and Phage strain information, b, Primers used in this study and c, Plasmids which were constructed. (XLSX 19 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Bondy-Denomy, J., Pawluk, A., Maxwell, K. et al. Bacteriophage genes that inactivate the CRISPR/Cas bacterial immune system. Nature 493, 429–432 (2013).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing