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. 2013 Jan 17;493(7432):429-32.
doi: 10.1038/nature11723. Epub 2012 Dec 16.

Bacteriophage genes that inactivate the CRISPR/Cas bacterial immune system

Joe Bondy-Denomy  1 , April Pawluk, Karen L Maxwell, Alan R Davidson
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Bacteriophage genes that inactivate the CRISPR/Cas bacterial immune system

Joe Bondy-Denomy et al. Nature. .
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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) genes. Similar to RNA interference in eukaryotes, these CRISPR/Cas systems use small RNAs for sequence-specific detection and neutralization of invading genomes. 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.

Figures

Figure 1. The CRISPR/Cas system is inhibited by expression of phage genes
a, Ten-fold dilutions of lysates of a CRISPR-sensitive phage (JBD18) and a CRISPR-insensitive phage (DMS3) were applied to bacterial lawns of wild-type (WT) PA14, PA14 lysogens (JBD24, MP29, or JBD30), and PA14 lacking a CRISPR/Cas system (ΔCR/cas). b, A schematic of the PA14 CRISPR loci and cas gene region is shown. Expanded versions of each CRISPR locus indicate the number of spacers in each, shown with white boxes, each of which is flanked by repeats denoted by black boxes. Black arrows indicate the CRISPR spacers corresponding to protospacers tested in Fig. 1c and gray arrows indicate the CRISPR spacers corresponding to protospacers tested in Supplementary Fig. 11. The DNA sequences of the protospacers tested in Fig. 1c are shown. c, Plasmids containing protospacers shown in Fig. 1b were electroporated into the indicated strains. The relative transformation efficiency was calculated by comparison with the transformation efficiency of the cloning vector containing no protospacer insert. Error bars represent standard deviation from the mean of three biological replicates. d, The anti-CRISPR genes of the indicated phages are located in the head gene regions of these genomes between genes homologous to the G gene of E. coli phage Mu (black boxes) and genes encoding the protease/scaffold protein of the phage head (gray boxes; see Supplementary Fig. 2). The percent identity of the proteins encoded by these genes to representatives from JBD30 are shown. The coloured boxes represent putative anti-CRISPR genes. Boxes of the same colour represent closely related genes and the sequence identity of their encoded proteins is indicated. Genes found to mediate anti-CRISPR activity are indicated by check marks and genes tested but displaying no anti-CRISPR activity are marked with an “X”. Unmarked genes were not tested due to their high similarity to tested genes. The gene box sizes are proportional to the sizes of the proteins in question (scale bar is 50 amino acids), but the spacing of the genes is not to scale. e, The same phages from Fig. 1a were tested on PA14 containing empty vector or plasmids expressing the indicated anti-CRISPR genes. Induction of the plasmid promoter with arabinose was required to produce a maximal effect for some of the anti-CRISPR genes as indicated by underlining. The assays shown in Fig. 1a,e are by necessity taken from different plates. The complete plates for these experiments and experiments with the other CRISPR-sensitive phages are shown in Supplementary Fig. 1.
Figure 2. An anti-CRISPR gene protects phages from the CRISPR/Cas system during infection
a, Ten-fold dilutions of lysates of anti-CRISPR phage JBD30, and the same phage with a frameshift mutation introduced into the anti-CRISPR gene 35 (gene 35fs) were applied to lawns of PA14 or PA14 ΔCR/cas. This experiment was carried out in a manner similar to those shown in Fig. 1a. b, A schematic representation of the in vivo homologous recombination between phage DMS3m and the anti-CRISPR region from phage JBD30. The X marks approximate the mapped region of recombination, up- and downstream of the anti-CRISPR gene 35 from JBD30 with details shown in Supplementary Fig. 12 and Methods. c, Ten-fold dilutions of lysates of a CRISPR-insensitive phage (DMS3), a CRISPR-sensitive phage (DMS3m), and DMS3m with anti-CRISPR gene 35 from JBD30 inserted (DMS3m::gene 35) were applied to lawns of PA14 or PA14 ΔCR/cas. d, A plasmid containing a protospacer matching CR1_sp1 (shown in Fig. 1b) was electroporated into the indicated lysogens or parent strain. As indicated, the prophages within these lysogens contain either a wild-type (WT) version of anti-CRISPR gene 35, a frameshift mutant of this gene, or no anti-CRISPR gene.
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Comment in

  • Phage biology: Giving CRISPR the slip.
    Kåhrström CT. Kåhrström CT. Nat Rev Microbiol. 2013 Feb;11(2):72. doi: 10.1038/nrmicro2958. Epub 2013 Jan 3. Nat Rev Microbiol. 2013. PMID: 23268230 No abstract available.

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