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Non-specific degradation of transcripts promotes plasmid clearance during type III-A CRISPR–Cas immunity

Nature Microbiology volume 4, pages 656–662 (2019)Cite this article

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Abstract

Type III-A CRISPR–Cas systems employ the Cas10–Csm complex to destroy bacteriophages and plasmids, using a guide RNA to locate complementary RNA molecules from the invader and trigger an immune response that eliminates the infecting DNA. In addition, these systems possess the non-specific RNase Csm6, which provides further protection for the host. While the role of Csm6 in immunity during phage infection has been determined, how this RNase is used against plasmids is unclear. Here, we show that Staphylococcus epidermidis Csm6 is required for immunity when transcription across the plasmid target is infrequent, leading to impaired target recognition and inefficient DNA degradation by the Cas10–Csm complex. In these conditions, Csm6 causes growth arrest in the host and prevents further plasmid replication through the indiscriminate degradation of host and plasmid transcripts. In contrast, when plasmid target sequences are efficiently transcribed, Csm6 is dispensable and DNA degradation by Cas10 is sufficient for anti-plasmid immunity. Csm6 therefore provides robustness to the type III-A CRISPR–Cas immune response against difficult targets for the Cas10–Csm complex.

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Fig. 1: Csm6 is required for interference against pG0400 when the target is weakly transcribed.
Fig. 2: Csm6 accelerates plasmid clearance when interfering against a weakly transcribed protospacer.
Fig. 3: Csm6 activation results in non-specific degradation of host and plasmid transcripts.
Fig. 4: Prevention of expression of genes important for plasmid replication accelerates plasmid clearance.
Fig. 5: The Cas10 HD domain is required for efficient plasmid clearance during type III-A immunity.

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Data availability

The data from this study are available from the authors upon request. The raw data for the RNA-seq experiments can be found at the Sequence Read Archive (NIH) through accession code PRJNA506073. Original gel pictures and northern blots are provided in Supplementary Fig. 6.

References

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

    Article  CAS  PubMed  Google Scholar 

  2. Marraffini, L. A. & Sontheimer, E. J. CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science 322, 1843–1845 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Gasiunas, G., Barrangou, R., Horvath, P. & Siksnys, V. Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc. Natl Acad. Sci. USA 109, E2579–E2586 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Hale, C. R. et al. RNA-guided RNA cleavage by a CRISPR RNA–Cas protein complex. Cell 139, 945–956 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Koonin, E. V., Makarova, K. S. & Zhang, F. Diversity, classification and evolution of CRISPR–Cas systems. Curr. Opin. Microbiol. 37, 67–78 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Pyenson, N. C. & Marraffini, L. A. Type III CRISPR–Cas systems: when DNA cleavage just isn’t enough. Curr. Opin. Microbiol. 37, 150–154 (2017).

    Article  CAS  PubMed  Google Scholar 

  9. Staals, R. H. et al. RNA targeting by the type III-A CRISPR–cas csm complex of Thermus thermophilus. Mol. Cell 56, 518–530 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Tamulaitis, G. et al. Programmable RNA shredding by the Type III-A CRISPR–Cas system of Streptococcus thermophilus. Mol. Cell 56, 506–517 (2014).

    Article  CAS  PubMed  Google Scholar 

  11. Zhang, J. et al. Structure and mechanism of the CMR complex for CRISPR-mediated antiviral immunity. Mol. Cell 45, 303–313 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Elmore, J. R. et al. Bipartite recognition of target RNAs activates DNA cleavage by the Type III-B CRISPR–Cas system. Genes Dev. 30, 447–459 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Estrella, M. A., Kuo, F. T. & Bailey, S. RNA-activated DNA cleavage by the Type III-B CRISPR–Cas effector complex. Genes Dev. 30, 460–470 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Kazlauskiene, M., Tamulaitis, G., Kostiuk, G., Venclovas, C. & Siksnys, V. Spatiotemporal control of type III-A CRISPR–Cas immunity: coupling DNA degradation with the target RNA recognition. Mol. Cell 62, 295–306 (2016).

    Article  CAS  PubMed  Google Scholar 

  15. Samai, P. et al. Co-transcriptional DNA and RNA cleavage during type III CRISPR–Cas immunity. Cell 161, 1164–1174 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Makarova, K. S. et al. An updated evolutionary classification of CRISPR–Cas systems. Nat. Rev. Microbiol. 13, 722–736 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Niewoehner, O. & Jinek, M. Structural basis for the endoribonuclease activity of the type III-A CRISPR-associated protein Csm6. RNA 22, 318–329 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Burroughs, A. M., Zhang, D., Schaffer, D. E., Iyer, L. M. & Aravind, L. Comparative genomic analyses reveal a vast, novel network of nucleotide-centric systems in biological conflicts, immunity and signaling. Nucleic Acids Res. 43, 10633–10654 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Kazlauskiene, M., Kostiuk, G., Venclovas, C., Tamulaitis, G. & Siksnys, V. A cyclic oligonucleotide signaling pathway in type III CRISPR–Cas systems. Science 357, 605–609 (2017).

    Article  CAS  PubMed  Google Scholar 

  20. Niewoehner, O. et al. Type III CRISPR–Cas systems produce cyclic oligoadenylate second messengers. Nature 548, 543–548 (2017).

    Article  CAS  PubMed  Google Scholar 

  21. Rouillon, C., Athukoralage, J. S., Graham, S., Gruschow, S. & White, M. F. Control of cyclic oligoadenylate synthesis in a type III CRISPR system. eLife 7, e36734 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  22. Athukoralage, J. S., Rouillon, C., Graham, S., Gruschow, S. & White, M. F. Ring nucleases deactivate type III CRISPR ribonucleases by degrading cyclic oligoadenylate. Nature 562, 277–280 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Jiang, W., Samai, P. & Marraffini, L. A. Degradation of phage transcripts by CRISPR-associated RNases enables type III CRISPR–Cas immunity. Cell 164, 710–721 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Foster, K., Kalter, J., Woodside, W., Terns, R. M. & Terns, M. P. The ribonuclease activity of Csm6 is required for anti-plasmid immunity by Type III-A CRISPR–Cas systems. RNA Biol. https://doi.org/10.1080/15476286.2018.1493334 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  25. Hatoum-Aslan, A., Maniv, I., Samai, P. & Marraffini, L. A. Genetic characterization of antiplasmid immunity through a type III-A CRISPR–CAS system. J. Bacteriol. 196, 310–317 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  26. Anantharaman, V., Iyer, L. M. & Aravind, L. Presence of a classical RRM-fold palm domain in Thg1-type 3′–5′nucleic acid polymerases and the origin of the GGDEF and CRISPR polymerase domains. Biol. Direct. 5, 43 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Deng, L., Garrett, R. A., Shah, S. A., Peng, X. & She, Q. A novel interference mechanism by a type IIIB CRISPR-Cmr module in Sulfolobus. Mol. Microbiol. 87, 1088–1099 (2013).

    Article  CAS  PubMed  Google Scholar 

  29. Goldberg, G. W., Jiang, W., Bikard, D. & Marraffini, L. A. Conditional tolerance of temperate phages via transcription-dependent CRISPR–Cas targeting. Nature 514, 633–637 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Kreiswirth, B. N. et al. The toxic shock syndrome exotoxin structural gene is not detectably transmitted by a prophage. Nature 305, 709–712 (1983).

    Article  CAS  PubMed  Google Scholar 

  31. Helle, L. et al. Vectors for improved Tet repressor-dependent gradual gene induction or silencing in Staphylococcus aureus. Microbiology 157, 3314–3323 (2011).

    Article  CAS  PubMed  Google Scholar 

  32. Liu, T. Y., Iavarone, A. T. & Doudna, J. A. RNA and DNA targeting by a reconstituted Thermus thermophilus Type III-A CRISPR–Cas system. PLoS ONE 12, e0170552 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  33. Ruiz-Maso, J. A. et al. Plasmid rolling-circle replication. Microbiol Spectr. 3, PLAS-0035-2014 (2015).

    Article  PubMed  Google Scholar 

  34. Sheppard, N. F., Glover, C. V. 3rd, Terns, R. M. & Terns, M. P. The CRISPR-associated Csx1 protein of Pyrococcus furiosus is an adenosine-specific endoribonuclease. RNA 22, 216–224 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Anantharaman, V., Makarova, K. S., Burroughs, A. M., Koonin, E. V. & Aravind, L. Comprehensive analysis of the HEPN superfamily: identification of novel roles in intra-genomic conflicts, defense, pathogenesis and RNA processing. Biol. Direct. 8, 15 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Upton, J. W. & Chan, F. K. Staying alive: cell death in antiviral immunity. Mol. Cell 54, 273–280 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  38. Horinouchi, S. & Weisblum, B. Nucleotide sequence and functional map of pE194, a plasmid that specifies inducible resistance to macrolide, lincosamide, and streptogramin type B antibodies. J. Bacteriol. 150, 804–814 (1982).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Horinouchi, S. & Weisblum, B. Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance. J. Bacteriol. 150, 815–825 (1982).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).

    Article  CAS  PubMed  Google Scholar 

  41. Ray, M. D., Boundy, S. & Archer, G. L. Transfer of the methicillin resistance genomic island among staphylococci by conjugation. Mol. Microbiol. 100, 675–685 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Lamberte, L. E. et al. Horizontally acquired AT-rich genes in Escherichia coli cause toxicity by sequestering RNA polymerase. Nat. Microbiol. 2, 16249 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Liao, Y., Smyth, G. K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930 (2014).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

The authors would like to thank A. Meeske and C. Mo for critical reading of the manuscript. They also thank the following: G. Goldberg for plasmid pGG25; C. Kenney and W. Jiang for sharing their insights on spc1-flip conjugation; the Rockefeller University Genomics Resource Center for performing the Csm6-targeting Nextseq RNA-seq experiment; and T. Carroll (of the Rockefeller University Bioinformatics Resource Center) and E. Stoyanova for helpful discussions on the bioinformatic analysis. J.T.R. was supported by a Boehringer Ingelheim Fonds Ph.D. fellowship. L.M. is supported by a Burroughs Wellcome Fund PATH Award and a NIH Director’s Pioneer Award (DP1GM128184).

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Authors and Affiliations

  1. Laboratory of Bacteriology, The Rockefeller University, New York, NY, USA

    Jakob T. Rostøl & Luciano A. Marraffini

  2. Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA

    Luciano A. Marraffini

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J.T.R. and L.M. designed the study. J.T.R performed all experiments and analysed the next-generation sequencing data. J.T.R. and L.M. wrote the manuscript.

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Correspondence to Luciano A. Marraffini.

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L.M. is a cofounder and Scientific Advisory Board member of Intellia Therapeutics and a cofounder of Eligo Biosciences. J.T.R. declares no competing interests.

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Rostøl, J.T., Marraffini, L.A. Non-specific degradation of transcripts promotes plasmid clearance during type III-A CRISPR–Cas immunity. Nat Microbiol 4, 656–662 (2019). https://doi.org/10.1038/s41564-018-0353-x

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