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Bacteriophage genes that inactivate the CRISPR/Cas bacterial immune system

Abstract

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.

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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).

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Acknowledgements

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.

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Authors

Contributions

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.

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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)

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Bondy-Denomy, J., Pawluk, A., Maxwell, K. et al. Bacteriophage genes that inactivate the CRISPR/Cas bacterial immune system. Nature 493, 429–432 (2013). https://doi.org/10.1038/nature11723

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