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RNA-targeting CRISPR–Cas systems

Nature Reviews Microbiology volume 21, pages 21–34 (2023)Cite this article

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Abstract

CRISPR–Cas is a widespread adaptive immune system in bacteria and archaea that protects against viral infection by targeting specific invading nucleic acid sequences. Whereas some CRISPR–Cas systems sense and cleave viral DNA, type III and type VI CRISPR–Cas systems sense RNA that results from viral transcription and perhaps invasion by RNA viruses. The sequence-specific detection of viral RNA evokes a cell-wide response that typically involves global damage to halt the infection. How can one make sense of an immune strategy that encompasses broad, collateral effects rather than specific, targeted destruction? In this Review, we summarize the current understanding of RNA-targeting CRISPR–Cas systems. We detail the composition and properties of type III and type VI systems, outline the cellular defence processes that are instigated upon viral RNA sensing and describe the biological rationale behind the broad RNA-activated immune responses as an effective strategy to combat viral infection.

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Fig. 1: DNA-targeting and RNA-targeting host defence systems and viral evasion strategies.
Fig. 2: Type III CRISPR–Cas immunity.
Fig. 3: Type VI CRISPR–Cas immunity.
Fig. 4: Model of the dormancy strategy used by type III and type VI CRISPR–Cas systems.
Fig. 5: Blindfolded self/non-self discrimination.

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References

  1. Koonin, E. V., Wolf, Y. I. & Katsnelson, M. I. Inevitability of the emergence and persistence of genetic parasites caused by evolutionary instability of parasite-free states. Biol. Direct 12, 1–12 (2017).

    Article  Google Scholar 

  2. Frost, L. S., Leplae, R., Summers, A. O. & Toussaint, A. Mobile genetic elements: The agents of open source evolution. Nat. Rev. Microbiol. 3, 722–732 (2005).

    Article  CAS  Google Scholar 

  3. Van Valen, L. A new evolutionary law. Evol. Theory 1, 1–30 (1973).

    Google Scholar 

  4. Koonin, E. V. Viruses and mobile elements as drivers of evolutionary transitions. Philos. Trans. R. Soc. B Biol. Sci. 371, 20150442 (2016).

    Article  Google Scholar 

  5. Bernheim, A. & Sorek, R. The pan-immune system of bacteria: antiviral defence as a community resource. Nat. Rev. Microbiol. 18, 113–119 (2020).

    Article  CAS  Google Scholar 

  6. Tesson, F. et al. Systematic and quantitative view of the antiviral arsenal of prokaryotes. Nat. Commun. 13, 2561 (2022).

    Article  CAS  Google Scholar 

  7. Rocha, E. P. C. & Bikard, D. Microbial defenses against mobile genetic elements and viruses: who defends whom from what? PLoS Biol. 20, e3001514 (2022).

    Article  CAS  Google Scholar 

  8. Hampton, H. G., Watson, B. N. J. & Fineran, P. C. The arms race between bacteria and their phage foes. Nature 577, 327–336 (2020).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  10. Deveau, H. et al. Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. J. Bacteriol. 190, 1390–1400 (2008).

    Article  CAS  Google Scholar 

  11. Barrangou, R. & Horvath, P. A decade of discovery: CRISPR functions and applications. Nat. Microbiol. 2, 17092 (2017).

    Article  CAS  Google Scholar 

  12. Datsenko, K. A. et al. Molecular memory of prior infections activates the CRISPR/Cas adaptive bacterial immunity system. Nat. Commun. 3, 1–7 (2012).

    Article  Google Scholar 

  13. Swarts, D. C., Mosterd, C., van Passel, M. W. J. & Brouns, S. J. J. CRISPR interference directs strand specific spacer acquisition. PLoS ONE 7, e35888 (2012).

    Article  CAS  Google Scholar 

  14. Nussenzweig, P. M., McGinn, J. & Marraffini, L. A. Cas9 cleavage of viral genomes primes the acquisition of new immunological memories. Cell Host Microbe 26, 515–526.e6 (2019).

    Article  CAS  Google Scholar 

  15. Carte, J., Wang, R., Li, H., Terns, R. M. & Terns, M. P. Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes. Genes Dev. 22, 3489–3496 (2008).

    Article  CAS  Google Scholar 

  16. East-Seletsky, A. et al. Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection. Nature 538, 270–273 (2016).

    Article  CAS  Google Scholar 

  17. Brouns, S. J. J. et al. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321, 960–964 (2008).

    Article  CAS  Google Scholar 

  18. Hille, F. et al. The biology of CRISPR-Cas: backward and forward. Cell 172, 1239–1259 (2018).

    Article  CAS  Google Scholar 

  19. Bernheim, A., Bikard, D., Touchon, M. & Rocha, E. P. C. Atypical organizations and epistatic interactions of CRISPRs and cas clusters in genomes and their mobile genetic elements. Nucleic Acids Res. 48, 748–760 (2020).

    CAS  Google Scholar 

  20. Jia, N. & Patel, D. J. Structure-based functional mechanisms and biotechnology applications of anti-CRISPR proteins. Nat. Rev. Mol. Cell Biol. 22, 563–579 (2021).

    Article  CAS  Google Scholar 

  21. Wilkinson, M. et al. Structural basis for the inhibition of RecBCD by Gam and its synergistic antibacterial effect with quinolones. Elife 5, 1–12 (2016).

    Article  Google Scholar 

  22. Shah, M. et al. A phage-encoded anti-activator inhibits quorum sensing in Pseudomonas aeruginosa. Mol. Cell 81, 571–583.e6 (2021).

    Article  CAS  Google Scholar 

  23. Bandyopadhyay, P. K., Studier, F. W., Hamilton, D. L. & Yuan, R. Inhibition of the type I restriction-modification enzymes EcoB and EcoK by the gene 0.3 protein of bacteriophage T7. J. Mol. Biol. 182, 567–578 (1985).

    Article  CAS  Google Scholar 

  24. Deveau, H., Garneau, J. E. & Moineau, S. CRISPR/Cas system and its role in phage-bacteria interactions. Annu. Rev. Microbiol. 64, 475–493 (2010).

    Article  CAS  Google Scholar 

  25. Hutinet, G., Lee, Y., de Crécy-lagard, V. & Weigele, P. R. Hypermodified DNA in viruses of E. coli and Salmonella. EcoSalPlus 9, eESP00282019 (2021).

    Google Scholar 

  26. Bryson, A. L. et al. Covalent modification of bacteriophage T4 DNA inhibits CRISPRCas9. mBio https://doi.org/10.1128/mBio.00648-15 (2015).

    Article  Google Scholar 

  27. Vlot, M. et al. Bacteriophage DNA glucosylation impairs target DNA binding by type I and II but not by type V CRISPR–Cas effector complexes. Nucleic Acids Res. 46, 873–885 (2018).

    Article  CAS  Google Scholar 

  28. Mendoza, S. D. et al. A bacteriophage nucleus-like compartment shields DNA from CRISPR nucleases. Nature 577, 244–248 (2020).

    Article  CAS  Google Scholar 

  29. Malone, L. M. et al. A jumbo phage that forms a nucleus-like structure evades CRISPR–Cas DNA targeting but is vulnerable to type III RNA-based immunity. Nat. Microbiol. 5, 48–55 (2020).

    Article  CAS  Google Scholar 

  30. 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  Google Scholar 

  31. Lopatina, A., Tal, N. & Sorek, R. Abortive infection: bacterial suicide as an antiviral immune strategy. Annu. Rev. Virol. 7, 371–384 (2020).

    Article  CAS  Google Scholar 

  32. Neri, U. et al. A five-fold expansion of the global RNA virome reveals multiple new clades of RNA bacteriophages. bioRxiv https://doi.org/10.1101/2022.02.15.480533 (2022).

    Article  Google Scholar 

  33. Wolf, Y. I. et al. Doubling of the known set of RNA viruses by metagenomic analysis of an aquatic virome. Nat. Microbiol. 5, 1262–1270 (2020).

    Article  CAS  Google Scholar 

  34. Artamonova, D. et al. Spacer acquisition by type III CRISPR–Cas system during bacteriophage infection of Thermus thermophilus. Nucleic Acids Res. 48, 9787–9803 (2020).

    Article  CAS  Google Scholar 

  35. Maniv, I., Jiang, W., Bikard, D. & Marraffini, L. A. Impact of different target sequences on type III CRISPR-Cas immunity. J. Bacteriol. 198, 941–950 (2016).

    Article  CAS  Google Scholar 

  36. Manica, A., Zebec, Z., Steinkellner, J. & Schleper, C. Unexpectedly broad target recognition of the CRISPR-mediated virus defence system in the archaeon Sulfolobus solfataricus. Nucleic Acids Res. 41, 10509–10517 (2013).

    Article  CAS  Google Scholar 

  37. Johnson, K., Learn, B. A., Estrella, M. A. & Bailey, S. Target sequence requirements of a type III-B CRISPR-Cas immune system. J. Biol. Chem. 294, 10290–10299 (2019).

    Article  CAS  Google Scholar 

  38. Pyenson, N. C., Gayvert, K., Varble, A., Elemento, O. & Marraffini, L. A. Broad targeting specificity during bacterial type III CRISPR-Cas immunity constrains viral escape. Cell Host Microbe 22, 343–353.e3 (2017).

    Article  CAS  Google Scholar 

  39. Gleditzsch, D. et al. PAM identification by CRISPR-Cas effector complexes: diversified mechanisms and structures. RNA Biol. 16, 504–517 (2019).

    Article  Google Scholar 

  40. Meeske, A. J. & Marraffini, L. A. RNA guide complementarity prevents self-targeting in type VI CRISPR systems. Mol. Cell 71, 791–801.e3 (2018).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  42. 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  Google Scholar 

  43. Vink, J. N. A., Baijens, J. H. L. & Brouns, S. J. J. PAM-repeat associations and spacer selection preferences in single and co-occurring CRISPR-Cas systems. Genome Biol. 22, 1–25 (2021).

    Article  Google Scholar 

  44. Majumdar, S. et al. Three CRISPR-Cas immune effector complexes coexist in Pyrococcus furiosus. RNA 21, 1147–1158 (2015).

    Article  CAS  Google Scholar 

  45. Silas, S. et al. Type III CRISPR-Cas systems can provide redundancy to counteract viral escape from type I systems. Elife 6, e27601 (2017).

    Article  Google Scholar 

  46. Hoikkala, V. et al. Cooperation between different CRISPR-Cas types enables adaptation in an RNA-targeting system. mBio 12, e03338-20 (2021).

    Article  Google Scholar 

  47. Chaikeeratisak, V. et al. Assembly of a nucleus-like structure during viral replication in bacteria. Science 355, 194–197 (2017).

    Article  CAS  Google Scholar 

  48. Harrison, E. & Brockhurst, M. A. Ecological and evolutionary benefits of temperate phage: what does or doesn’t kill you makes you stronger. Bioessays 39, 1700112 (2017).

    Article  Google Scholar 

  49. 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  Google Scholar 

  50. Goldberg, G. W. et al. Incomplete prophage tolerance by type III-A CRISPR-Cas systems reduces the fitness of lysogenic hosts. Nat. Commun. 9, 61 (2018).

    Article  Google Scholar 

  51. Koonin, E. V. & Makarova, K. S. Discovery of oligonucleotide signaling mediated by CRISPR-associated polymerases solves two puzzles but leaves an enigma. ACS Chem. Biol. 13, 309–312 (2018).

    Article  CAS  Google Scholar 

  52. Coleman, G. A. et al. A rooted phylogeny resolves early bacterial evolution. Science. 372, (2021).

  53. Makarova, K. S. et al. Evolutionary classification of CRISPR–Cas systems: a burst of class 2 and derived variants. Nat. Rev. Microbiol. 18, 67–83 (2020). Presents the latest evolutionary classification of CRISPR–Cas systems and cas genes.

    Article  CAS  Google Scholar 

  54. Molina, R., Sofos, N. & Montoya, G. Structural basis of CRISPR-Cas type III prokaryotic defence systems. Curr. Opin. Struct. Biol. 65, 119–129 (2020). Summarizes the structural insights into the mechanisms governing type III CRISPR–Cas immunity.

    Article  CAS  Google Scholar 

  55. Tamulaitis, G. Venclovas, Č. & Siksnys, V. Type III CRISPR-Cas immunity: major differences brushed aside. Trends Microbiol. 25, 49–61 (2017).

    Article  CAS  Google Scholar 

  56. Rouillon, C. et al. Structure of the CRISPR interference complex CSM reveals key similarities with cascade. Mol. Cell 52, 124–134 (2013).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  58. Hale, C. R. et al. RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex. Cell 139, 945–956 (2009). Describes the type III CRISPR–Cas effector for the first time.

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  60. Grüschow, S., Adamson, C. S. & White, M. F. Specificity and sensitivity of an RNA targeting type III CRISPR complex coupled with a NucC endonuclease effector. Nucleic Acids Res. 49, 13122–13134 (2021).

    Article  Google Scholar 

  61. Sokolowski, R. D., Graham, S. & White, M. F. Cas6 specificity and CRISPR RNA loading in a complex CRISPR-Cas system. Nucleic Acids Res. 42, 6532–6541 (2014).

    Article  CAS  Google Scholar 

  62. Walker, F. C., Chou-Zheng, L., Dunkle, J. A. & Hatoum-Aslan, A. Molecular determinants for CRISPR RNA maturation in the Cas10-Csm complex and roles for non-Cas nucleases. Nucleic Acids Res. 45, 2112–2123 (2017).

    CAS  Google Scholar 

  63. Chou-Zheng, L. & Hatoum-Aslan, A. A type III-A CRISPR-Cas system employs degradosome nucleases to ensure robust immunity. Elife 8, 1–25 (2019).

    Article  Google Scholar 

  64. van Beljouw, S. P. B. et al. The gRAMP CRISPR-Cas effector is an RNA endonuclease complexed with a caspase-like peptidase. Science 3, eabk2718 (2021).

    Google Scholar 

  65. Özcan, A. et al. Programmable RNA targeting with the single-protein CRISPR effector Cas7-11. Nature 597, 720–725 (2021).

    Article  Google Scholar 

  66. 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  Google Scholar 

  67. Kazlauskiene, M., Tamulaitis, G., Kostiuk, G., Venclovas, Č. & 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  Google Scholar 

  68. Steens, J. A. et al. SCOPE enables type III CRISPR-Cas diagnostics using flexible targeting and stringent CARF ribonuclease activation. Nat. Commun. 12, 1–12 (2021).

    Article  Google Scholar 

  69. 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  Google Scholar 

  70. Wang, L. et al. Dynamics of Cas10 govern discrimination between self and non-self in type III CRISPR-Cas immunity. Mol. Cell 73, 278–290.e4 (2019).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  72. Jia, N. et al. Type III-A CRISPR-Cas Csm complexes: assembly, periodic RNA cleavage, DNase activity regulation, and autoimmunity. Mol. Cell 73, 264–277.e5 (2019).

    Article  CAS  Google Scholar 

  73. Staals, R. H. J. et al. Structure and activity of the RNA-targeting type III-B CRISPR-Cas complex of Thermus thermophilus. Mol. Cell 52, 135–145 (2013).

    Article  CAS  Google Scholar 

  74. Hale, C. R., Cocozaki, A., Li, H., Terns, R. M. & Terns, M. P. Target RNA capture and cleavage by the Cmr type III-B CRISPR–Cas effector complex. Genes Dev. 28, 2432–2443 (2014).

    Article  Google Scholar 

  75. Hale, C. R. et al. Essential features and rational design of CRISPR RNAs that function with the Cas RAMP module complex to cleave RNAs. Mol. Cell 45, 292–302 (2012).

    Article  CAS  Google Scholar 

  76. Rouillon, C., Athukoralage, J. S., Graham, S., Grüschow, S. & White, M. F. Control of cyclic oligoadenylate synthesis in a type III CRISPR system. Elife 7, 1–22 (2018).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  78. Park, K.-H. et al. RNA activation-independent DNA targeting of the type III CRISPR-Cas system by a Csm complex. EMBO Rep. 18, 826–840 (2017).

    Article  CAS  Google Scholar 

  79. Liu, T. Y., Liu, J. J., Aditham, A. J., Nogales, E. & Doudna, J. A. Target preference of type III-A CRISPR-Cas complexes at the transcription bubble. Nat. Commun. 10, 3001 (2019).

    Article  Google Scholar 

  80. Mo, C. Y. et al. Type III-A CRISPR immunity promotes mutagenesis of staphylococci. Nature 592, 611–615 (2021).

    Article  CAS  Google Scholar 

  81. 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).

    Article  Google Scholar 

  82. Han, W. et al. A Type III-B Cmr effector complex catalyzes the synthesis of cyclic oligoadenylate second messengers by cooperative substrate binding. Nucleic Acids Res. 46, 10319–10330 (2018).

    CAS  Google Scholar 

  83. Cocozaki, A. I. et al. Structure of the Cmr2 subunit of the CRISPR-Cas RNA silencing complex. Structure 20, 545–553 (2012).

    Article  CAS  Google Scholar 

  84. Nasef, M. et al. Regulation of cyclic oligoadenylate synthesis by the Staphylococcus epidermidis Cas10-Csm complex. RNA 25, 948–962 (2019).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  86. Niewoehner, O. et al. Type III CRISPR-Cas systems produce cyclic oligoadenylate second messengers. Nature 548, 543–548 (2017). Describes the discovery of cOA-based signalling upon RNA activation in type III CRISPR–Cas systems.

    Article  CAS  Google Scholar 

  87. Shah, S. A. et al. Comprehensive search for accessory proteins encoded with archaeal and bacterial type III CRISPR-cas gene cassettes reveals 39 new cas gene families. RNA Biol. 16, 530–542 (2019). Identifies a vast range of protein families associated with type III CRISPR–Cas systems.

    Article  Google Scholar 

  88. Makarova, K. S. et al. Evolutionary and functional classification of the CARF domain superfamily, key sensors in prokaryotic antivirus defense. Nucleic Acids Res. 48, 8828–8847 (2020). Identifies a vast range of protein families associated with type III CRISPR–Cas systems.

    Article  CAS  Google Scholar 

  89. Makarova, K. S., Anantharaman, V., Grishin, N. V., Koonin, E. V. & Aravind, L. CARF and WYL domains: Ligand-binding regulators of prokaryotic defense systems. Front. Genet. 5, 102 (2014).

    Article  Google Scholar 

  90. Garcia-Doval, C. et al. Activation and self-inactivation mechanisms of the cyclic oligoadenylate-dependent CRISPR ribonuclease Csm6. Nat. Commun. 11, 1596 (2020).

    Article  CAS  Google Scholar 

  91. 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  Google Scholar 

  92. Han, W., Pan, S., López-Méndez, B., Montoya, G. & She, Q. Allosteric regulation of Csx1, a type IIIB-associated CARF domain ribonuclease by RNAs carrying a tetraadenylate tail. Nucleic Acids Res. 45, 10740–10750 (2017).

    Article  CAS  Google Scholar 

  93. Zhao, Y. et al. Structural insights into the CRISPR-Cas-associated ribonuclease activity of Staphylococcus epidermidis Csm3 and Csm6. Sci. Bull. 63, 691–699 (2018).

    Article  CAS  Google Scholar 

  94. McMahon, S. A. et al. Structure and mechanism of a type III CRISPR defence DNA nuclease activated by cyclic oligoadenylate. Nat. Commun. 11, 500 (2020).

    Article  CAS  Google Scholar 

  95. Zhu, W. et al. The CRISPR ancillary effector Can2 is a dual-specificity nuclease potentiating type III CRISPR defence. Nucleic Acids Res. 49, 2777–2789 (2021).

    Article  CAS  Google Scholar 

  96. Rostøl, J. T. et al. The Card1 nuclease provides defence during type-III CRISPR immunity. Nature 590, 624–629 (2021).

    Article  Google Scholar 

  97. Athukoralage, J. S., Graham, S., Grüschow, S., Rouillon, C. & White, M. F. A type III CRISPR ancillary ribonuclease degrades its cyclic oligoadenylate activator. J. Mol. Biol. 431, 2894–2899 (2019).

    Article  CAS  Google Scholar 

  98. Hinton, D. M. Transcriptional control in the prereplicative phase of T4 development. Virol. J. 7, 1–16 (2010).

    Article  Google Scholar 

  99. Chevallereau, A. et al. Next-generation “-omics” approaches reveal a massive alteration of host RNA metabolism during bacteriophage infection of Pseudomonas aeruginosa. PLoS Genet. 12, 1–20 (2016).

    Article  Google Scholar 

  100. Blasdel, B. G., Chevallereau, A., Monot, M., Lavigne, R. & Debarbieux, L. Comparative transcriptomics analyses reveal the conservation of an ancestral infectious strategy in two bacteriophage genera. ISME J. 11, 1988–1996 (2017).

    Article  CAS  Google Scholar 

  101. Nemudraia, A. et al. Sequence-specific capture and concentration of viral RNA by type III CRISPR system enhances diagnostic. Res. Sq. https://doi.org/10.21203/rs.3.rs-1466718/v1 (2022).

    Article  Google Scholar 

  102. Smalakyte, D. et al. Type III-A CRISPR-associated protein Csm6 degrades cyclic hexa-adenylate activator using both CARF and HEPN domains. Nucleic Acids Res. 48, 9204–9217 (2020).

    Article  CAS  Google Scholar 

  103. Grüschow, S., Adamson, C. S. & White, M. F. Specificity and sensitivity of an RNA targeting type III CRISPR complex coupled with a NucC endonuclease effector. 49 13122–13134 (2021).

  104. Lau, R. K. et al. Structure and mechanism of a cyclic trinucleotide-activated bacterial endonuclease mediating bacteriophage immunity. Mol. Cell 77, 723–733.e6 (2020).

    Article  CAS  Google Scholar 

  105. Mayo-Muñoz, D. et al. Type III CRISPR–Cas provides resistance against nucleus-forming jumbo phages via abortive infection. bioRxiv https://doi.org/10.1101/2022.06.20.496707 (2022).

    Article  Google Scholar 

  106. Burroughs, A. M., Zhang, D., Schäffer, 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  Google Scholar 

  107. Lowey, B. et al. CBASS immunity uses CARF-related effectors to sense 3′–5′- and 2′–5′-linked cyclic oligonucleotide signals and protect bacteria from phage infection. Cell 182, 38–49.e17 (2020).

    Article  CAS  Google Scholar 

  108. Rouillon, C. et al. SAVED by a toxin: structure and function of the CRISPR Lon protease. bioRxiv https://doi.org/10.1101/2021.12.06.471393 (2021).

    Article  Google Scholar 

  109. Athukoralage, J. S. et al. The dynamic interplay of host and viral enzymes in type III CRISPR-mediated cyclic nucleotide signalling. Elife 9, 1–16 (2020).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  111. Brown, S., Gauvin, C. C., Charbonneau, A. A., Burman, N. & Martin Lawrence, C. Csx3 is a cyclic oligonucleotide phosphodiesterase associated with type III CRISPR-Cas that degrades the second messenger cA4. J. Biol. Chem. 295, 14963–14972 (2020).

    Article  CAS  Google Scholar 

  112. Foster, K., Grüschow, S., Bailey, S., White, M. F. & Terns, M. P. Regulation of the RNA and DNA nuclease activities required for Pyrococcus furiosus type III-B CRISPR-Cas immunity. Nucleic Acids Res. 48, 4418–4434 (2020).

    Article  CAS  Google Scholar 

  113. Jia, N., Jones, R., Yang, G., Ouerfelli, O. & Patel, D. J. CRISPR-Cas III-A Csm6 CARF domain is a ring nuclease triggering stepwise cA4 cleavage with ApA>p formation terminating RNase activity. Mol. Cell 75, 944–956.e6 (2019).

    Article  CAS  Google Scholar 

  114. Samolygo, A., Athukoralage, J. S., Graham, S. & White, M. F. Fuse to defuse: a self-limiting ribonuclease-ring nuclease fusion for type III CRISPR defence. Nucleic Acids Res. 48, 6149–6156 (2020).

    Article  CAS  Google Scholar 

  115. Athukoralage, J. S. & White, M. F. Cyclic oligoadenylate signaling and regulation by ring nucleases during type III CRISPR defense. RNA 27, 855–867 (2021).

    Article  CAS  Google Scholar 

  116. Zhao, R. et al. A membrane-associated DHH-DHHA1 nuclease degrades type III CRISPR second messenger. Cell Rep. 32, 108133 (2020).

    Article  CAS  Google Scholar 

  117. Athukoralage, J. S. et al. An anti-CRISPR viral ring nuclease subverts type III CRISPR immunity. Nature 577, 572–575 (2020).

    Article  CAS  Google Scholar 

  118. Charbonneau, A. A., Eckert, D. M., Gauvin, C. C., Lintner, N. G. & Lawrence, C. M. Cyclic tetra-adenylate (cA4) recognition by Csa3; implications for an integrated Class 1 CRISPR-Cas immune response in Saccharolobus solfataricus. Biomolecules 11, 1852 (2021).

    Article  CAS  Google Scholar 

  119. Ye, Q. et al. CRISPR-associated factor Csa3b regulates CRISPR adaptation and Cmr-mediated RNA interference in Sulfolobus islandicus. Front. Microbiol. 11, 1–12 (2020).

    Article  Google Scholar 

  120. Xia, P., Dutta, A., Gupta, K., Batish, M. & Parashar, V. Structural basis of cyclic oligoadenylate binding to the transcription factor Csa3 outlines cross talk between type III and type I CRISPR systems. J. Biol. Chem. 298, 101591 (2022).

    Article  CAS  Google Scholar 

  121. Zhang, S. et al. Mycobacterium tuberculosis CRISPR/Cas system Csm1 holds clues to the evolutionary relationship between DNA polymerase and cyclase activity. Int. J. Biol. Macromol. 170, 140–149 (2021).

    Article  CAS  Google Scholar 

  122. Liu, L. et al. Two distant catalytic sites are responsible for C2c2 RNase activities. Cell 168, 121–134.e12 (2017).

    Article  CAS  Google Scholar 

  123. Liu, L. et al. The molecular architecture for RNA-guided RNA cleavage by Cas13a. Cell 170, 714–726.e10 (2017).

    Article  CAS  Google Scholar 

  124. Abudayyeh, O. O. et al. C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science 353, aaf5573 (2016). Characterizes the effector protein Cas13 (then known as C2c2) for the first time.

    Article  Google Scholar 

  125. Xu, C. et al. Programmable RNA editing with compact CRISPR–Cas13 systems from uncultivated microbes. Nat. Methods 18, 499–506 (2021).

    Article  CAS  Google Scholar 

  126. Yan, W. X. et al. Cas13d is a compact RNA-targeting type VI CRISPR effector positively modulated by a WYL-domain-containing accessory protein. Mol. Cell 70, 327–339.e5 (2018).

    Article  CAS  Google Scholar 

  127. Smargon, A. A. et al. Cas13b is a type VI-B CRISPR-associated RNA-guided RNase differentially regulated by accessory proteins Csx27 and Csx28. Mol. Cell 65, 618–630.e7 (2017).

    Article  CAS  Google Scholar 

  128. Gootenberg, J. S. et al. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science 356, 438–442 (2017).

    Article  CAS  Google Scholar 

  129. Tambe, A., East-Seletsky, A., Knott, G. J., Doudna, J. A. & O’Connell, M. R. RNA Binding and HEPN-nuclease activation are decoupled in CRISPR-Cas13a. Cell Rep. 24, 1025–1036 (2018).

    Article  CAS  Google Scholar 

  130. Meeske, A. J., Nakandakari-Higa, S. & Marraffini, L. A. Cas13-induced cellular dormancy prevents the rise of CRISPR-resistant bacteriophage. Nature 570, 241–245 (2019). Demonstrates that the ancillary nuclease activity of Cas13 halts host growth to abort viral infection.

    Article  CAS  Google Scholar 

  131. Adler, B. A. et al. RNA-targeting CRISPR-Cas13 provides broad-spectrum phage immunity. bioRxiv https://doi.org/10.1101/2022.03.25.485874 (2022).

    Article  Google Scholar 

  132. Meeske, A. J. et al. A phage-encoded anti-CRISPR enables complete evasion of type VI-A CRISPR-Cas immunity. Science 369, 54–59 (2020).

    Article  CAS  Google Scholar 

  133. VanderWal, A. R., Park, J.-U., Polevoda, B., Kellogg, E. H. & O’Connell, M. R. CRISPR-Csx28 forms a Cas13b-activated membrane pore required for robust CRISPR-Cas adaptive immunity. bioRxiv https://doi.org/10.1101/2021.11.02.466367 (2021).

    Article  Google Scholar 

  134. Guan, J., Bosch, A. O., Mendoza, S. D., Karambelkar, S. & Berry, J. RNA targeting with CRISPR-Cas13a facilitates bacteriophage genome engineering. bioRxiv https://doi.org/10.1101/2022.02.14.480438 (2022).

    Article  Google Scholar 

  135. Bandaru, S. et al. Structure-based design of gRNA for Cas13. Sci. Rep. 10, 1–12 (2020).

    Article  Google Scholar 

  136. Wang, B. et al. Structural basis for self-cleavage prevention by tag:anti-tag pairing complementarity in type VI Cas13 CRISPR systems. Mol. Cell 81, 1100–1115.e5 (2021).

    Article  CAS  Google Scholar 

  137. O’Connell, M. R. Molecular mechanisms of RNA targeting by Cas13-containing type VI CRISPR–Cas systems. J. Mol. Biol. 431, 66–87 (2019). Evaluates the structural insights into the architecture and mechanisms of type VI CRISPR–Cas effectors.

    Article  Google Scholar 

  138. East-Seletsky, A., O’Connell, M. R., Burstein, D., Knott, G. J. & Doudna, J. A. RNA targeting by functionally orthogonal type VI-A CRISPR-Cas enzymes. Mol. Cell 66, 373–383.e3 (2017).

    Article  CAS  Google Scholar 

  139. Gootenberg, J. S. et al. Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a and Csm6. Science 360, 439–444 (2018).

    Article  CAS  Google Scholar 

  140. Williams, M. C. et al. Phage genome cleavage enables resuscitation from Cas13 induced bacterial dormancy. bioRxiv https://doi.org/10.1101/2022.07.05.498905 (2022).

    Article  Google Scholar 

  141. Jain, I. et al. tRNA anticodon cleavage by target-activated CRISPR-Cas13a effector. bioRxiv https://doi.org/10.1101/2021.11.10.468108 (2021).

    Article  Google Scholar 

  142. Su, Z., Wilson, B., Kumar, P. & Dutta, A. Noncanonical roles of tRNAs: tRNA fragments and beyond. Annu. Rev. Genet. 54, 47–69 (2020).

    Article  CAS  Google Scholar 

  143. Makarova, K. S., Gao, L., Zhang, F. & Koonin, E. V. Unexpected connections between type VI-B CRISPR-Cas systems, bacterial natural competence, ubiquitin signaling network and DNA modification through a distinct family of membrane proteins. FEMS Microbiol. Lett. 366, 1–6 (2019).

    Article  Google Scholar 

  144. Shivram, H., Cress, B. F., Knott, G. J. & Doudna, J. A. Controlling and enhancing CRISPR systems. Nat. Chem. Biol. 17, 10–19 (2021).

    Article  CAS  Google Scholar 

  145. Zhang, H., Dong, C., Li, L., Wasney, G. A. & Min, J. Structural insights into the modulatory role of the accessory protein WYL1 in the type VI-D CRISPR-Cas system. Nucleic Acids Res. 47, 5420–5428 (2019).

    Article  CAS  Google Scholar 

  146. Rittershaus, E. S. C., Baek, S. H. & Sassetti, C. M. The normalcy of dormancy: common themes in microbial quiescence. Cell Host Microbe 13, 643–651 (2013).

    Article  CAS  Google Scholar 

  147. Dworkin, J. & Shah, I. M. Exit from dormancy in microbial organisms. Nat. Rev. Microbiol. 8, 890–896 (2010).

    Article  CAS  Google Scholar 

  148. Abudayyeh, O. O. et al. RNA targeting with CRISPR-Cas13. Nature 550, 280–284 (2017).

    Article  Google Scholar 

  149. Koonin, E. V. & Zhang, F. Coupling immunity and programmed cell suicide in prokaryotes: life-or-death choices. BioEssays 39, 1–9 (2017). Provides the conceptual basis on how viral immunity in bacteria and archaea is related to programmed cell death.

    Article  CAS  Google Scholar 

  150. Makarova, K. S., Anantharaman, V., Aravind, L. & Koonin, E. V. Live virus-free or die: coupling of antivirus immunity and programmed suicide or dormancy in prokaryotes. Biol. Direct 7, 1–10 (2012).

    Article  Google Scholar 

  151. Mendoza, S. D. & Bondy-Denomy, J. Cas13 helps bacteria play dead when the enemy strikes. Cell Host Microbe 26, 1–2 (2019).

    Article  CAS  Google Scholar 

  152. Weigel, C. & Seitz, H. Bacteriophage replication modules. FEMS Microbiol. Rev. 30, 321–381 (2006).

    Article  CAS  Google Scholar 

  153. Muñoz-Espín, D., Serrano-Heras, G. & Salas, M. Role of Host Factors in Bacteriophage φ29 DNA Replication. in Advances in Virus Research vol. 82 351–383 (Academic Press Inc., 2012).

  154. Chevallereau, A., Pons, B. J., van Houte, S. & Westra, E. R. Interactions between bacterial and phage communities in natural environments. Nat. Rev. Microbiol. 20, 49–62 (2022).

    Article  CAS  Google Scholar 

  155. Vink, J. N. A. et al. Direct visualization of native CRISPR target search in live bacteria reveals cascade DNA surveillance mechanism. Mol. Cell 77, 39–50.e10 (2020).

    Article  CAS  Google Scholar 

  156. Dimitriu, T. et al. Bacteriostatic antibiotics promote CRISPR-Cas adaptive immunity by enabling increased spacer acquisition. Cell Host Microbe 30, 31–40.e5 (2022).

    Article  CAS  Google Scholar 

  157. de Jonge, P. A., Nobrega, F. L., Brouns, S. J. J. & Dutilh, B. E. Molecular and evolutionary determinants of bacteriophage host range. Trends Microbiol. 27, 51–63 (2019).

    Article  Google Scholar 

  158. Ndhlovu, A., Durand, P. M. & Ramsey, G. Programmed cell death as a black queen in microbial communities. Mol. Ecol. 30, 1110–1119 (2021).

    Article  CAS  Google Scholar 

  159. Peeters, S. H. & de Jonge, M. I. For the greater good: programmed cell death in bacterial communities. Microbiol. Res. 207, 161–169 (2018).

    Article  CAS  Google Scholar 

  160. Weigele, P. & Raleigh, E. A. Biosynthesis and function of modified bases in bacteria and their viruses. Chem. Rev. 116, 12655–12687 (2016).

    Article  CAS  Google Scholar 

  161. Leenay, R. T. & Beisel, C. L. Deciphering, communicating, and engineering the CRISPR PAM. J. Mol. Biol. 429, 177–191 (2017).

    Article  CAS  Google Scholar 

  162. Shmakov, S. et al. Diversity and evolution of class 2 CRISPR-Cas systems. Nat. Rev. Microbiol. 15, 169–182 (2017).

    Article  CAS  Google Scholar 

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Acknowledgements

S.J.J.B. is supported by the Netherlands Organization for Scientific Research (VICI grant no. VI.C.182.027) and has received funding from the European Research Council Consolidator Grant programme under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 101003229). The authors thank members of the Brouns laboratory for helpful discussions.

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Authors and Affiliations

  1. Department of Bionanoscience, Delft University of Technology, Delft, Netherlands

    Sam P. B. van Beljouw, Jasper Sanders, Alicia Rodríguez-Molina & Stan J. J. Brouns

  2. Kavli Institute of Nanoscience, Delft, Netherlands

    Sam P. B. van Beljouw, Jasper Sanders, Alicia Rodríguez-Molina & Stan J. J. Brouns

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Contributions

S.P.B.v.B., J.S., A.R.-M. and S.J.J.B. researched data for the article and contributed substantially to discussion of the content. S.P.B.v.B. wrote the manuscript. S.P.B.v.B. and A.R.-M. designed figures. S.P.B.v.B. and S.J.J.B. reviewed and edited the manuscript before submission.

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Glossary

Mobile genetic elements

(MGEs). Simple genetic organisms (such as phages, plasmids, conjugative elements, transposons, introns and phage-inducible chromosomal islands) capable of horizontal transfer within or between genomes.

Dormancy

A non-replicating cellular state defined by low or inactive metabolism that promotes survival during stressful conditions.

Protospacer adjacent motif

(PAM). A small DNA sequence which serves as a recognition motif for various CRISPR–Cas nucleases, positioned next to a target sequence in an invading DNA-based mobile genetic element but absent from the host genome, allowing the discrimination between self and non-self.

Protospacer flanking site

(PFS). A small RNA sequence positioned next to a target sequence in RNA which needs to be non-complementary to the repeat-derived portion of the CRISPR RNA for the type III and type VI CRISPR–Cas effectors to become active, thereby preventing activation by a self-transcript.

CRISPR adaptation

The process by which a sequence of an invading mobile genetic element is extracted and stored in the CRISPR array.

Palm domain

A domain in the Cas10 subunit of various type III CRISPR–Cas effectors with structural similarity to nucleotidyl cyclases and nucleotide polymerases, catalysing the synthesis of cyclic oligoadenylate molecules from ATP.

Cyclic oligonucleotide-based antiphage signalling system

A family of defence systems in bacteria and archaea related to animal cGAS–STING immunity in which a cyclase generates cyclic oligonucleotides to activate a cell death effector protein in response to viral infection.

Restriction–modification systems

Genetic compositions encoding nucleases and methylases in which specific methylation patterns generated by the methylases prevent or facilitate cleavage by the nucleases, presenting a means for discriminating self from non-self.

Membrane depolarization

The process by which the basal electrochemical potential across cellular membranes changes from a negative state to a positive state.

Toxin–antitoxin systems

Genetic compositions encoding at least a toxin and a counteracting antitoxin whereby the toxin component becomes active when the antitoxin is absent.

Kin selection

A theory of natural selection describing behaviour that favours the fitness of closely related kin over the fitness of the individual enacting the behaviour.

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van Beljouw, S.P.B., Sanders, J., Rodríguez-Molina, A. et al. RNA-targeting CRISPR–Cas systems. Nat Rev Microbiol 21, 21–34 (2023). https://doi.org/10.1038/s41579-022-00793-y

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