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.

  • Protocol
  • Published:

Detecting natural adaptation of the Streptococcus thermophilus CRISPR-Cas systems in research and classroom settings

Nature Protocols volume 12, pages 547–565 (2017)Cite this article

Subjects

Abstract

CRISPR (clustered regularly interspaced short palindromic repeats)-Cas systems have been adapted into a powerful genome-editing tool. The basis for the flexibility of the tool lies in the adaptive nature of CRISPR-Cas as a bacterial immune system. Here, we describe a protocol to experimentally demonstrate the adaptive nature of this bacterial immune system by challenging the model organism for the study of CRISPR adaptation, Streptococcus thermophilus, with phages in order to detect natural CRISPR immunization. A bacterial culture is challenged with lytic phages, the surviving cells are screened by PCR for expansion of their CRISPR array and the newly acquired specificities are mapped to the genome of the phage. Furthermore, we offer three variants of the assay to (i) promote adaptation by challenging the system using defective viruses, (ii) challenge the system using plasmids to generate plasmid-resistant strains and (iii) bias the system to obtain natural immunity against a specifically targeted DNA sequence. The core protocol and its variants serve as a means to explore CRISPR adaptation, discover new CRISPR-Cas systems and generate bacterial strains that are resistant to phages or refractory to undesired genes or plasmids. In addition, the core protocol has served in teaching laboratories at the undergraduate level, demonstrating both its robust nature and educational value. Carrying out the core protocol takes 4 h of hands-on time over 7 d. Unlike sequence-based methods for detecting natural CRISPR adaptation, this phage-challenge-based approach results in the isolation of CRISPR-immune bacteria for downstream characterization and use.

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: The repeat–spacer structure of the CRISPR loci CR1 and CR3 as they appear in the genome of S. thermophilus DGCC7710.
Figure 2: Schematic depiction of the core protocol and the steps it shares in common with its three variants.
Figure 3: Schematic depiction of the initial steps of Variant 2 (Boxes 3 and 4), beginning with the plasmid-containing strain (green circle) and ending where it rejoins the core protocol (Fig. 2).
Figure 4
Figure 5: Generation of BIMs.
Figure 6: Verification of spacer acquisition by PCR.
Figure 7: Flowchart of the undergraduate laboratory sessions.

Similar content being viewed by others

Accession codes

Accessions

NCBI Reference Sequence

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

    Article  CAS  PubMed  Google Scholar 

  3. Deltcheva, E. et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471, 602–607 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Garneau, J.E. et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468, 67–71 (2010).

    Article  CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Jiang, W., Bikard, D., Cox, D., Zhang, F. & Marraffini, L.A. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat. Biotechnol. 31, 233–239 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Ran, F.A. et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Larson, M.H. et al. CRISPR interference (CRISPRi) for sequence-specific control of gene expression. Nat. Protoc. 8, 2180–2196 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. 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 

  10. Yosef, I., Goren, M.G. & Qimron, U. Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli. Nucleic Acids Res. 12, 5569–5576 (2012).

    Article  Google Scholar 

  11. Wei, Y., Chesne, M.T., Terns, R.M. & Terns, M.P. Sequences spanning the leader-repeat junction mediate CRISPR adaptation to phage in Streptococcus thermophilus. Nucleic Acids Res. 43, 1749–1758 (2015).

    Article  CAS  PubMed  PubMed Central  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  CAS  PubMed  Google Scholar 

  13. Samson, J.E. & Moineau, S. Bacteriophages in food fermentations: new frontiers in a continuous arms race. Annu. Rev. Food Sci. Technol. 4, 347–368 (2013).

    Article  CAS  PubMed  Google Scholar 

  14. Hynes, A.P., Villion, M. & Moineau, S. Adaptation in bacterial CRISPR-Cas immunity can be driven by defective phages. Nat. Commun. 5, 4399 (2014).

    Article  CAS  PubMed  Google Scholar 

  15. 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  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Hynes, A.P., Labrie, S.J. & Moineau, S. Programming native CRISPR arrays for the generation of targeted immunity. MBio 7, e00202–e00216 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Díez-Villaseñor, C., Guzmán, N.M., Almendros, C., García-Martínez, J. & Mojica, F.J.M. CRISPR-spacer integration reporter plasmids reveal distinct genuine acquisition specificities among CRISPR-Cas I-E variants of Escherichia coli. RNA Biol. 10, 792–802 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  19. 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  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  PubMed  Google Scholar 

  21. Shmakov, S. et al. Pervasive generation of oppositely oriented spacers during CRISPR adaptation. Nucleic Acids Res. 42, 5907–5916 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Pride, D.T. et al. Analysis of streptococcal CRISPRs from human saliva reveals substantial sequence diversity within and between subjects over time. Genome Res. 21, 126–136 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Lopez-Sanchez, M.J. et al. The highly dynamic CRISPR1 system of Streptococcus agalactiae controls the diversity of its mobilome. Mol. Microbiol. 85, 1057–1071 (2012).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  25. Bondy-Denomy, J., Pawluk, A., Maxwell, K.L. & Davidson, A.R. Bacteriophage genes that inactivate the CRISPR/Cas bacterial immune system. Nature 493, 429–432 (2013).

    Article  CAS  PubMed  Google Scholar 

  26. Martel, B. & Moineau, S. CRISPR-Cas: an efficient tool for genome engineering of virulent bacteriophages. Nucleic Acids Res. 42, 9504–9513 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Magadán, A.H., Dupuis, M.-È., Villion, M. & Moineau, S. Cleavage of phage DNA by the Streptococcus thermophilus CRISPR3-Cas system. PLoS One 7, e40913 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  28. van der Ploeg, J.R. Analysis of CRISPR in Streptococcus mutans suggests frequent occurrence of acquired immunity against infection by M102-like bacteriophages. Microbiology 155, 1966–1976 (2009).

    Article  CAS  PubMed  Google Scholar 

  29. Cady, K.C., Bondy-Denomy, J., Heussler, G.E., Davidson, A.R. & O'Toole, G.A. The CRISPR/Cas adaptive immune system of Pseudomonas aeruginosa mediates resistance to naturally occurring and engineered Phages. J. Bacteriol. 194, 5728–5738 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. 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  CAS  PubMed  PubMed Central  Google Scholar 

  31. 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  PubMed  PubMed Central  Google Scholar 

  32. Erdmann, S. & Garrett, R.A. Selective and hyperactive uptake of foreign DNA by adaptive immune systems of an archaeon via two distinct mechanisms. Mol. Microbiol. 85, 1044–1056 (2012).

    Article  CAS  PubMed  PubMed Central  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  CAS  PubMed  Google Scholar 

  34. Hooton, S.P.T. & Connerton, I.F. Campylobacter jejuni acquire new host-derived CRISPR spacers when in association with bacteriophages harboring a CRISPR-like Cas4 protein. Front. Microbiol. 5, 744 (2014).

    PubMed  Google Scholar 

  35. Erdmann, S., Le Moine Bauer, S. & Garrett, R.A. Inter-viral conflicts that exploit host CRISPR immune systems of Sulfolobus. Mol. Microbiol. 91, 900–917 (2014).

    Article  CAS  PubMed  Google Scholar 

  36. Heler, R. et al. Cas9 specifies functional viral targets during CRISPR-Cas adaptation. Nature 519, 199–202 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Rao, C. et al. Active and adaptive Legionella CRISPR-Cas reveals a recurrent challenge to the pathogen. Cell. Microbiol. 18, 1319–38 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Silas, S. et al. Direct CRISPR spacer acquisition from RNA by a natural reverse transcriptase–Cas1 fusion protein. Science 351, 929–932 (2016).

    Article  Google Scholar 

Download references

Acknowledgements

We recognize members of our team who were involved in the initial development of these protocols, namely M.-È. Dupuis, J. Garneau, S. Labrie, A. Magadán, B. Martel and M. Villion. M. Sabri was involved in the initial implementation of the protocol at the undergraduate level. We thank A. Renaud for assistance with materials to generate figures.

A.P.H. is supported by a scholarship from the National Science and Engineering Research Council of Canada (NSERC). M.-L.L. is supported by scholarships from the Fonds de Recherche du Québec—Nature et Technologies (FRQNT), Novalait and Op+Lait. S.M. acknowledges funding from the Natural Sciences and Engineering Research Council of Canada (Discovery program), Canadian Institutes of Health Research (Team Grant–Emerging: Novel Alternatives to Antibiotics) and Danisco/DuPont. S.M. holds a T1 Canada Research Chair in Bacteriophages.

Author information

Authors and Affiliations

  1. Département de biochimie, de microbiologie et de bio-informatique, Faculté des sciences et de génie,

    Alexander P Hynes, Marie-Laurence Lemay, Luc Trudel, Hélène Deveau, Michel Frenette, Denise M Tremblay & Sylvain Moineau

  2. Université Laval, Québec, Québec, Canada

    Alexander P Hynes, Marie-Laurence Lemay, Luc Trudel, Hélène Deveau, Michel Frenette, Denise M Tremblay & Sylvain Moineau

  3. Groupe de recherche en écologie buccale, Faculté de médecine dentaire, Université Laval, Québec, Québec, Canada

    Alexander P Hynes, Marie-Laurence Lemay, Michel Frenette, Denise M Tremblay & Sylvain Moineau

  4. Félix d'Hérelle Reference Center for Bacterial Viruses, Faculté de médecine dentaire, Université Laval, Québec, Québec, Canada

    Denise M Tremblay & Sylvain Moineau

Authors
  1. Alexander P Hynes
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Marie-Laurence Lemay
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Luc Trudel
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hélène Deveau
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Michel Frenette
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Denise M Tremblay
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Sylvain Moineau
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.P.H. developed Variants 1 and 3. M.-L.L. was in the first undergraduate cohort to perform the protocols, and aided in their implementation in the following 2 years. L.T. and M.F. implemented the core protocol in undergraduate laboratories in all 3 years. H.D. helped develop the initial core protocol, and implemented it in undergraduate laboratories for 2 years. S.M. and D.M.T. were involved in the development of the core protocol and the three variants, and implemented the core protocol in undergraduate laboratories in the first year. All authors contributed to the writing of the manuscript.

Corresponding author

Correspondence to Sylvain Moineau.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Hynes, A., Lemay, ML., Trudel, L. et al. Detecting natural adaptation of the Streptococcus thermophilus CRISPR-Cas systems in research and classroom settings. Nat Protoc 12, 547–565 (2017). https://doi.org/10.1038/nprot.2016.186

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2016.186

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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