Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Anti-cas spacers in orphan CRISPR4 arrays prevent uptake of active CRISPR–Cas I-F systems

Nature Microbiology volume 1, Article number: 16081 (2016) Cite this article

Subjects

Abstract

Archaea and bacteria harbour clustered regularly interspaced short palindromic repeats (CRISPR) loci. These arrays encode RNA molecules (crRNA), each containing a sequence of a single repeat–intervening spacer. The crRNAs guide CRISPR-associated (Cas) proteins to cleave nucleic acids complementary to the crRNA spacer, thus interfering with targeted foreign elements. Notably, pre-existing spacers may trigger the acquisition of new spacers from the target molecule by means of a primed adaptation mechanism. Here, we show that naturally occurring orphan CRISPR arrays that contain spacers matching sequences of the cognate (absent) cas genes are able to elicit both primed adaptation and direct interference against genetic elements carrying those genes. Our findings show the existence of an anti-cas mechanism that prevents the transfer of a fully equipped CRISPR–Cas system. Hence, they suggest that CRISPR immunity may be undesired by particular prokaryotes, potentially because they could limit possibilities for gaining favourable sequences by lateral transfer.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Diversity of CRISPR4.1–2 in E. coli.
Figure 2: Plasmid loss assays in BL21 AI.
Figure 3: Coverage of spacers acquired in BL21 AI + pCas1–3 + pCas5–8 grown without antibiotic selection.
Figure 4: Coverage of spacers acquired under antibiotic selection.

Similar content being viewed by others

References

  1. Grissa, I., Vergnaud, G. & Pourcel, C. The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats. BMC Bioinformatics 8, 172 (2007).

    Article  Google Scholar 

  2. Mojica, F. J. M. & Garrett, R. A. in CRISPR-Cas Systems: RNA-mediated Adaptive Immunity in Bacteria and Archaea (eds Barrangou, R. & van der Oost, J. ) 1–31 (Springer, 2013).

    Book  Google Scholar 

  3. Mojica, F. J., Díez-Villaseñor, C., Soria, E. & Juez, G. Biological significance of a family of regularly spaced repeats in the genomes of Archaea, bacteria and mitochondria. Mol. Microbiol. 36, 244–246 (2000).

    Article  Google Scholar 

  4. Jansen, R., Embden, J. D., Gaastra, W. & Schouls, L. M. Identification of genes that are associated with DNA repeats in prokaryotes. Mol. Microbiol. 43, 1565–1575 (2002).

    Article  Google Scholar 

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

    Article  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Van der Oost, J., Westra, E. R., Jackson, R. N. & Wiedenheft, B. Unravelling the structural and mechanistic basis of CRISPR-Cas systems. Nature Rev. Microbiol. 12, 479–492 (2014).

    Article  Google Scholar 

  8. Westra, E. R. et al. The CRISPRs, they are a-changin’: how prokaryotes generate adaptive immunity. Annu. Rev. Genet. 46, 311–339 (2012).

    Article  Google Scholar 

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

    Article  Google Scholar 

  10. Sinkunas, T. et al. Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system. EMBO J. 30, 1335–1342 (2011).

    Article  Google Scholar 

  11. 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  Google Scholar 

  12. Mojica, F. J. M., Díez-Villaseñor, C., García-Martínez, J. & Almendros, C. Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology 155, 733–740 (2009).

    Article  Google Scholar 

  13. Bolotin, A., Quinquis, B., Sorokin, A. & Ehrlich, S. D. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology 151, 2551–2561 (2005).

    Article  Google Scholar 

  14. Wiedenheft, B. et al. RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions. Proc. Natl Acad. Sci. USA 108, 10092–10097 (2011).

    Article  Google Scholar 

  15. Xue, C. et al. CRISPR interference and priming varies with individual spacer sequences. Nucleic Acids Res. 15, 10831–10847 (2015).

    Article  Google Scholar 

  16. Semenova, E. et al. Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence. Proc. Natl Acad. Sci. USA 108, 10098–10103 (2011).

    Article  Google Scholar 

  17. Vercoe, R. B. et al. Cytotoxic chromosomal targeting by CRISPR/Cas systems can reshape bacterial genomes and expel or remodel pathogenicity islands. PLoS Genet. 9, e1003454 (2013).

    Article  Google Scholar 

  18. Fineran, P. C. et al. Degenerate target sites mediate rapid primed CRISPR adaptation. Proc. Natl Acad. Sci. USA 111, E1629–E1638 (2014).

    Article  Google Scholar 

  19. Heler, R., Marraffini, L. A. & Bikard, D. Adapting to new threats: the generation of memory by CRISPR-Cas immune systems. Mol. Microbiol. 93, 1–9 (2014).

    Article  Google Scholar 

  20. Amitai, G. & Sorek, R. CRISPR-Cas adaptation: insights into the mechanism of action. Nature Rev. Microbiol. 14, 67–76 (2016).

    Article  Google Scholar 

  21. Nuñez, J. K., Lee, A. S., Engelman, A. & Doudna, J. A. Integrase-mediated spacer acquisition during CRISPR-Cas adaptive immunity. Nature 519, 193–198 (2015).

    Article  Google Scholar 

  22. Yosef, I., Goren, M. G. & Qimron, U. Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli. Nucleic Acids Res. 40, 5569–5576 (2012).

    Article  Google Scholar 

  23. Pourcel, C., Salvignol, G. & Vergnaud, G. CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology 151, 653–663 (2005).

    Article  Google Scholar 

  24. Mojica, F. J. M., Díez-Villaseñor, C., García-Martínez, J. & Soria, E. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J. Mol. Evol. 60, 174–182 (2005).

    Article  Google Scholar 

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

    Article  Google Scholar 

  26. Bult, C. J. et al. Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii. Science 273, 1058–1073 (1996).

    Article  Google Scholar 

  27. Lillestol, R. K., Redder, P., Garrett, R. A. & Brügger, K. A putative viral defence mechanism in archaeal cells. Archaea 2, 59–72 (2006).

    Article  Google Scholar 

  28. Pul, U. et al. Identification and characterization of E. coli CRISPR-cas promoters and their silencing by H-NS. Mol. Microbiol. 75, 1495–1512 (2010).

    Article  Google Scholar 

  29. Shah, S. A., Erdmann, S., Mojica, F. J. & Garrett, R. A. Protospacer recognition motifs: mixed identities and functional diversity. RNA Biol. 10, 891–899 (2013).

    Article  Google Scholar 

  30. Fineran, P. C. & Charpentier, E. Memory of viral infections by CRISPR-Cas adaptive immune systems: acquisition of new information. Virology 434, 202–209 (2012).

    Article  Google Scholar 

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

    Article  Google Scholar 

  32. Datsenko, K. A. et al. Molecular memory of prior infections activates the CRISPR/Cas adaptive bacterial immunity system. Nature Commun. 3, 945 (2012).

    Article  Google Scholar 

  33. Li, M., Wang, R., Zhao, D. & Xiang, H. Adaptation of the Haloarcula hispanica CRISPR-Cas system to a purified virus strictly requires a priming process. Nucleic Acids Res. 42, 2483–2492 (2014).

    Article  Google Scholar 

  34. Richter, C. et al. Priming in the Type I-F CRISPR-Cas system triggers strand-independent spacer acquisition, bi-directionally from the primed protospacer. Nucleic Acids Res. 42, 8516–8526 (2014).

    Article  Google Scholar 

  35. Sesto, N. et al. A PNPase dependent CRISPR System in Listeria. PLoS Genet. 10, e1004065 (2014).

    Article  Google Scholar 

  36. Touchon, M. & Rocha, E. P. The small, slow and specialized CRISPR and anti-CRISPR of Escherichia and Salmonella. PLoS ONE 5, e11126 (2010).

    Article  Google Scholar 

  37. Díez-Villaseñor, C., Almendros, C., García-Martínez, J. & Mojica, F. J. M. Diversity of CRISPR loci in Escherichia coli. Microbiology 156, 1351–1361 (2010).

    Article  Google Scholar 

  38. Kunin, V., Sorek, R. & Hugenholtz, P. Evolutionary conservation of sequence and secondary structures in CRISPR repeats. Genome Biol. 8, R61 (2007).

    Article  Google Scholar 

  39. Toro, M. et al. Association of clustered regularly interspaced short palindromic repeat (CRISPR) elements with specific serotypes and virulence potential of shiga toxin-producing Escherichia coli. Appl. Environ. Microbiol. 80, 1411–1420 (2014).

    Article  Google Scholar 

  40. Vorontsova, D. et al. Foreign DNA acquisition by the I-F CRISPR-Cas system requires all components of the interference machinery. Nucleic Acids Res. 43, 10848–10860 (2015).

    Article  Google Scholar 

  41. Almendros, C., Guzmán, N. M., Díez-Villaseñor, C., García-Martínez, J. & Mojica, F. J. M. Target motifs affecting natural immunity by a constitutive CRISPR-Cas system in Escherichia coli. PLoS ONE 7, e50797 (2012).

    Article  Google Scholar 

  42. Almendros, C., Mojica, F. J., Díez-Villaseñor, C., Guzmán, N. M. & García-Martínez, J. CRISPR-Cas functional module exchange in Escherichia coli. MBio 5, e00767 (2014).

    Article  Google Scholar 

  43. Levy, A. et al. CRISPR adaptation biases explain preference for acquisition of foreign DNA. Nature 520, 505–510 (2015).

    Article  Google Scholar 

  44. Selander, R. K. et al. Methods of multilocus enzyme electrophoresis for bacterial population genetics and systematics. Appl. Environ. Microbiol. 51, 873–884 (1986).

    Google Scholar 

  45. Seed, K. D., Lazinski, D. W., Calderwood, S. B. & Camilli, A. A bacteriophage encodes its own CRISPR/Cas adaptive response to evade host innate immunity. Nature 494, 489–491 (2013).

    Article  Google Scholar 

  46. Westra, E. R. et al. Type I-E CRISPR-cas systems discriminate target from non-target DNA through base pairing-independent PAM recognition. PLoS Genet. 9, e1003742 (2013).

    Article  Google Scholar 

  47. Delaney, N. F. et al. Ultrafast evolution and loss of CRISPRs following a host shift in a novel wildlife pathogen, Mycoplasma gallisepticum. PLoS Genet. 8, e1002511 (2012).

    Article  Google Scholar 

  48. Pougach, K. et al. Transcription, processing and function of CRISPR cassettes in Escherichia coli. Mol. Microbiol. 77, 1367–1379 (2010).

    Article  Google Scholar 

  49. Westra, E. R. et al. H-NS-mediated repression of CRISPR-based immunity in Escherichia coli K12 can be relieved by the transcription activator LeuO. Mol. Microbiol. 77, 1380–1393 (2010).

    Article  Google Scholar 

  50. García-Gutiérrez, E., Almendros, C., Mojica, F. J., Guzmán, N. M. & García-Martínez, J. CRISPR content correlates with the pathogenic potential of Escherichia coli. PLoS ONE 10, e0131935 (2015).

    Article  Google Scholar 

  51. Grissa, I., Vergnaud, G. & Pourcel, C. CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats. Nucleic Acids Res. 35, W52–W57 (2007).

    Article  Google Scholar 

  52. Grissa, I., Vergnaud, G. & Pourcel, C. CRISPRcompar: a website to compare clustered regularly interspaced short palindromic repeats. Nucleic Acids Res. 36, W145–W148 (2008).

    Article  Google Scholar 

  53. Biswas, A., Gagnon, J. N., Brouns, S. J., Fineran, P. C. & Brown, C. M. CRISPRTarget: bioinformatic prediction and analysis of crRNA targets. RNA Biol. 10, 817–827 (2013).

    Article  Google Scholar 

  54. Kearse, M. et al. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28, 1647–1649 (2012).

    Article  Google Scholar 

  55. Crooks, G. E., Hon, G., Chandonia, J. M. & Brenner, S. E. WebLogo: a sequence logo generator. Genome Res. 14, 1188–1190 (2004).

    Article  Google Scholar 

  56. Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl Acad. Sci. USA 97, 6640–6645 (2000).

    Article  Google Scholar 

  57. Wiedenheft, B. et al. Structural basis for DNase activity of a conserved protein implicated in CRISPR-mediated genome defense. Structure 17, 904–912 (2009).

    Article  Google Scholar 

  58. Shi, X. et al. Enhancing Escherichia coli electrotransformation competency by invoking physiological adaptations to stress and modifying membrane integrity. Anal. Biochem. 320, 152–155 (2003).

    Article  Google Scholar 

Download references

Acknowledgements

This work was supported by the Ministerio de Economía y Competitividad (BIO2011-24417 and BIO2014-53029-P) and the Consellería D'Educació, Cultura i Esport, Generalitat Valenciana (ACOMP/2014/135). The authors thank E. Denamur (INSERM U722-Université Paris Diderot, France) for E. coli strain ED1a.

Author information

Authors and Affiliations

  1. Departamento de Fisiología, Genética y Microbiología, Universidad de Alicante, Alicante, Spain

    Cristóbal Almendros, Noemí M. Guzmán, Jesús García-Martínez & Francisco J. M. Mojica

  2. Instituto Multidisciplinar para el Estudio del Medio ‘Ramón Margalef’, Universidad de Alicante, Alicante, Spain

    Francisco J. M. Mojica

Authors
  1. Cristóbal Almendros
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Noemí M. Guzmán
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jesús García-Martínez
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Francisco J. M. Mojica
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

C.A. and N.M.G. performed the experiments. C.A., J.G.-M. and F.J.M.M. conceived and designed the experiments. C.A. and F.J.M.M. analysed the data and wrote the paper, with comments from the co-authors.

Corresponding authors

Correspondence to Cristóbal Almendros or Francisco J. M. Mojica.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Figures 1-7 and Supplementary Tables 1-3 (PDF 10943 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Almendros, C., Guzmán, N., García-Martínez, J. et al. Anti-cas spacers in orphan CRISPR4 arrays prevent uptake of active CRISPR–Cas I-F systems. Nat Microbiol 1, 16081 (2016). https://doi.org/10.1038/nmicrobiol.2016.81

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/nmicrobiol.2016.81

This article is cited by

Search

Advanced search

Quick links

Nature Briefing Microbiology

Sign up for the Nature Briefing: Microbiology newsletter — what matters in microbiology research, free to your inbox weekly.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing: Microbiology