Protocol | Published:

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

Nature Protocols volume 12, pages 547565 (2017) | Download Citation

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.

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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

Affiliations

  1. Département de biochimie, de microbiologie et de bio-informatique, Faculté des sciences et de génie, Université Laval, Québec, Québec, Canada.

    • Alexander P Hynes
    • , Marie-Laurence Lemay
    • , Luc Trudel
    • , Hélène Deveau
    • , Michel Frenette
    • , Denise M Tremblay
    •  & Sylvain Moineau
  2. 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
  3. Félix d'Hérelle Reference Center for Bacterial Viruses, Faculté de médecine dentaire, Université Laval, Québec, Québec, Canada.

    • Denise M Tremblay
    •  & Sylvain Moineau

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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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Sylvain Moineau.

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DOI

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

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