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An anti-CRISPR from a virulent streptococcal phage inhibits Streptococcus pyogenes Cas9

Abstract

The CRISPR–Cas system owes its utility as a genome-editing tool to its origin as a prokaryotic immune system. The first demonstration of its activity against bacterial viruses (phages) is also the first record of phages evading that immunity1. This evasion can be due to point mutations1, large-scale deletions2, DNA modifications3, or phage-encoded proteins that interfere with the CRISPR–Cas system, known as anti-CRISPRs (Acrs)4. The latter are of biotechnological interest, as Acrs can serve as off switches for CRISPR-based genome editing5. Every Acr characterized to date originated from temperate phages, genomic islands, or prophages4,5,6,7,8, and shared properties with the first Acr discovered. Here, with a phage-oriented approach, we have identified an unrelated Acr in a virulent phage of Streptococcus thermophilus. In challenging a S. thermophilus strain CRISPR-immunized against a set of virulent phages, we found one that evaded the CRISPR-encoded immunity >40,000× more often than the others. Through systematic cloning of its genes, we identified an Acr solely responsible for the abolished immunity. We extended our findings by demonstrating activity in another S. thermophilus strain, against unrelated phages, and in another bacterial genus immunized using the heterologous SpCas9 system favoured for genome editing. This Acr completely abolishes SpCas9-mediated immunity in our assays.

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Fig. 1: Discovery of virulent phages impeding CRISPR-based immunity.
Fig. 2: Anti-CRISPR activity of AcrIIA5 in S. thermophilus.
Fig. 3: Anti-CRISPR gene and protein.
Fig. 4: Anti-CRISPR activity against SpCas9.

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Acknowledgements

The authors thank M.-C. Jodeau and I. Chavichvily for the initial technical work on the efficiency of spacer acquisition in S. thermophilus DGCC7854. A.P.H. is supported by an NSERC Postdoctoral Fellowships award. M.-L.L. is supported by scholarships from the Fonds de Recherche du Québec-Nature et Technologies, Novalait and Op+Lait. S.M. acknowledges funding from the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Program and DuPont. S.M. holds a Tier 1 Canada Research Chair in Bacteriophages.

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A.P.H. and S.M. jointly conceived the study. G.M.R., M.-L.L., P.H., D.A.R., and C.F. contributed technical guidance. A.P.H. designed all experiments performed in this study. A.P.H. and G.M.R. generated all subsequent data presented in the manuscript. M.-L.L. created plasmid constructs for the experiments presented in Fig. 4. A.P.H. wrote the manuscript. A.P.H. generated all associated figures. G.M.R., M.-L.L., P.H., D.A.R., C.F. and S.M commented on the manuscript.

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Correspondence to Sylvain Moineau.

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A.P.H., G.M.R., M.-L.L., P.H., D.A.R., C.F. and S.M. are co-inventors on patent(s) or patent application(s) related to CRISPR–Cas systems and their various uses.

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Supplementary Figure 1, Supplementary Table 1, Supplementary References.

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Hynes, A.P., Rousseau, G.M., Lemay, ML. et al. An anti-CRISPR from a virulent streptococcal phage inhibits Streptococcus pyogenes Cas9. Nat Microbiol 2, 1374–1380 (2017). https://doi.org/10.1038/s41564-017-0004-7

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