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Inactivation of CRISPR-Cas systems by anti-CRISPR proteins in diverse bacterial species


CRISPR-Cas systems provide sequence-specific adaptive immunity against foreign nucleic acids1,2. They are present in approximately half of all sequenced prokaryotes3 and are expected to constitute a major barrier to horizontal gene transfer. We previously described nine distinct families of proteins encoded in Pseudomonas phage genomes that inhibit CRISPR-Cas function4,5. We have developed a bioinformatic approach that enabled us to discover additional anti-CRISPR proteins encoded in phages and other mobile genetic elements of diverse bacterial species. We show that five previously undiscovered families of anti-CRISPRs inhibit the type I-F CRISPR-Cas systems of both Pseudomonas aeruginosa and Pectobacterium atrosepticum, and a dual specificity anti-CRISPR inactivates both type I-F and I-E CRISPR-Cas systems. Mirroring the distribution of the CRISPR-Cas systems they inactivate, these anti-CRISPRs were found in species distributed broadly across the phylum Proteobacteria. Importantly, anti-CRISPRs originating from species with divergent type I-F CRISPR-Cas systems were able to inhibit the two systems we tested, highlighting their broad specificity. These results suggest that all type I-F CRISPR-Cas systems are vulnerable to inhibition by anti-CRISPRs. Given the widespread occurrence and promiscuous activity of the anti-CRISPRs described here, we propose that anti-CRISPRs play an influential role in facilitating the movement of DNA between prokaryotes by breaching the barrier imposed by CRISPR-Cas systems.

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Figure 1: Discovery and characterization of aca1-associated anti-CRISPR genes.
Figure 2: Investigation of AcrF6Pae dual anti-CRISPR activity.
Figure 3: Discovery and characterization of aca2-associated anti-CRISPR genes.
Figure 4: Newly identified anti-CRISPRs impact diverse type I-F CRISPR-Cas systems.


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This work was supported by funding from the Canadian Institutes of Health Research to A.R.D. (MOP-130482) and K.L.M. (MOP-136845). A.P. was supported by an Ontario Graduate Scholarship and a CIHR Canada Graduate Scholarship Doctoral Award. R.H.J.S. was funded by a University of Otago Division of Health Sciences Career Development Post-doctoral Fellowship. P.C.F was supported by a Rutherford Discovery Fellowship from the Royal Society of New Zealand. B.N.J.W. was supported by a University of Otago Doctoral Scholarship.

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Authors and Affiliations



A.P. designed experiments, performed bioinformatic analysis, performed experiments, analysed data and wrote the manuscript. R.H.J.S. designed and performed Pectobacterium experiments and wrote parts of the manuscript. C.T. performed Pectobacterium experiments. B.N.J.W. performed Pectobacterium experiments and wrote parts of the manuscript. S.S. assembled and uploaded to NCBI the genome sequences of P. aeruginosa strains SMC4386 and SMC4386 ΔCRISPR-Cas. P.C.F. designed Pectobacterium experiments and wrote parts of the manuscript. K.L.M. supervised experiments and wrote the manuscript. A.R.D. supervised experiments and wrote the manuscript.

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Correspondence to Karen L. Maxwell or Alan R. Davidson.

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The authors declare no competing financial interests.

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Supplementary Figure 1, Supplementary Tables 1-6, Supplementary References. (PDF 514 kb)

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Pawluk, A., Staals, R., Taylor, C. et al. Inactivation of CRISPR-Cas systems by anti-CRISPR proteins in diverse bacterial species. Nat Microbiol 1, 16085 (2016).

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