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.

Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials

Subjects

Abstract

Antibiotics target conserved bacterial cellular pathways or growth functions and therefore cannot selectively kill specific members of a complex microbial population. Here, we develop programmable, sequence-specific antimicrobials using the RNA-guided nuclease Cas9 (refs.1,2) delivered by a bacteriophage. We show that Cas9, reprogrammed to target virulence genes, kills virulent, but not avirulent, Staphylococcus aureus. Reprogramming the nuclease to target antibiotic resistance genes destroys staphylococcal plasmids that harbor antibiotic resistance genes3,4 and immunizes avirulent staphylococci to prevent the spread of plasmid-borne resistance genes. We also show that CRISPR-Cas9 antimicrobials function in vivo to kill S. aureus in a mouse skin colonization model. This technology creates opportunities to manipulate complex bacterial populations in a sequence-specific manner.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: Sequence-specific killing of S. aureus by a phagemid-delivered CRISPR system.
Figure 2: Targeting antibiotic resistance genes and plasmids in an MRSA strain.
Figure 3: Sequence-specific killing of kanamycin-resistant S. aureus in a mouse skin colonization model.

References

  1. 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 

  2. 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 

  3. Diep, B.A. et al. Complete genome sequence of USA300, an epidemic clone of community-acquired meticillin-resistant Staphylococcus aureus. Lancet 367, 731–739 (2006).

    Article  CAS  PubMed  Google Scholar 

  4. Weigel, L.M. et al. Genetic analysis of a high-level vancomycin-resistant isolate of Staphylococcus aureus. Science 302, 1569–1571 (2003).

    Article  CAS  PubMed  Google Scholar 

  5. Round, J.L. et al. The Toll-like receptor 2 pathway establishes colonization by a commensal of the human microbiota. Science 332, 974–977 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Mazmanian, S.K., Round, J.L. & Kasper, D.L. A microbial symbiosis factor prevents intestinal inflammatory disease. Nature 453, 620–625 (2008).

    Article  CAS  PubMed  Google Scholar 

  7. Dethlefsen, L., Huse, S., Sogin, M.L. & Relman, D.A. The pervasive effects of an antibiotic on the human gut microbiota, as revealed by deep 16S rRNA sequencing. PLoS Biol. 6, e280 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  8. 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 

  9. Gomaa, A.A. et al. Programmable removal of bacterial strains by use of genome-targeting CRISPR-Cas systems. MBio 5, e00928–e00913 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  10. 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 

  11. Melnikov, A.A., Tchernov, A.P., Fodor, I. & Bayev, A.A. Lambda phagemids and their transducing properties. Gene 28, 29–35 (1984).

    Article  CAS  PubMed  Google Scholar 

  12. 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  CAS  PubMed  PubMed Central  Google Scholar 

  13. Grice, E.A. et al. Topographical and temporal diversity of the human skin microbiome. Science 324, 1190–1192 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Lowy, F.D. Staphylococcus aureus infections. N. Engl. J. Med. 339, 520–532 (1998).

    Article  CAS  PubMed  Google Scholar 

  15. 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 

  16. 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 

  17. Bikard, D., Hatoum-Aslan, A., Mucida, D. & Marraffini, L.A. CRISPR interference can prevent natural transformation and virulence acquisition during in vivo bacterial infection. Cell Host Microbe 12, 177–186 (2012).

    Article  CAS  PubMed  Google Scholar 

  18. Baba, T., Bae, T., Schneewind, O., Takeuchi, F. & Hiramatsu, K. Genome sequence of Staphylococcus aureus strain Newman and comparative analysis of staphylococcal genomes: polymorphism and evolution of two major pathogenicity islands. J. Bacteriol. 190, 300–310 (2008).

    Article  CAS  PubMed  Google Scholar 

  19. Charpentier, E. et al. Novel cassette-based shuttle vector system for gram-positive bacteria. Appl. Environ. Microbiol. 70, 6076–6085 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Ubukata, K., Nonoguchi, R., Matsuhashi, M. & Konno, M. Expression and inducibility in Staphylococcus aureus of the mecA gene, which encodes a methicillin-resistant S. aureus-specific penicillin-binding protein. J. Bacteriol. 171, 2882–2885 (1989).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Isnard, C., Malbruny, B., Leclercq, R. & Cattoir, V. Genetic basis for in vitro and in vivo resistance to lincosamides, streptogramins A, and pleuromutilins (LSAP Phenotype) in Enterococcus faecium. Antimicrob. Agents Chemother. 57, 4463–4469 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Kugelberg, E. et al. Establishment of a superficial skin infection model in mice by using Staphylococcus aureus and Streptococcus pyogenes. Antimicrob. Agents Chemother. 49, 3435–3441 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Pastagia, M. et al. A novel chimeric lysin shows superiority to mupirocin for skin decolonization of methicillin-resistant and -sensitive Staphylococcus aureus strains. Antimicrob. Agents Chemother. 55, 738–744 (2011).

    Article  CAS  PubMed  Google Scholar 

  24. Agarwal, R. & Roy, K. Intracellular delivery of polymeric nanocarriers: a matter of size, shape, charge, elasticity and surface composition. Ther. Deliv. 4, 705–723 (2013).

    Article  CAS  PubMed  Google Scholar 

  25. Bikard, D. et al. Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Res. 41, 7429–7437 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Qi, L.S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. McDougal, L.K. et al. Pulsed-field gel electrophoresis typing of oxacillin-resistant Staphylococcus aureus isolates from the United States: establishing a national database. J. Clin. Microbiol. 41, 5113–5120 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Bae, T., Baba, T., Hiramatsu, K. & Schneewind, O. Prophages of Staphylococcus aureus Newman and their contribution to virulence. Mol. Microbiol. 62, 1035–1047 (2006).

    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 10.1038/nature13637 (31 August 2014).

  30. Gibson, D.G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We would like to thank A. Raz for providing plasmid pCN57 and D. Mucida for assistance with flow cytometry experiments. D.B. is supported by the Bettencourt Schuller Foundation. L.A.M. is supported by the Searle Scholars Program, the Rita Allen Scholars Program, an Irma T. Hirschl Award, a Sinsheimer Foundation Award and a NIH Director's New Innovator Award (1DP2AI104556-01). V.A.F. is supported by NIH Grant AI057472.

Author information

Authors and Affiliations

Authors

Contributions

D.B. and L.A.M. designed the experiments. D.B. and W.J. performed the in vitro experiments. D.B., P.M.N. and C.W.E. performed the animal experiments. G.W.G. isolated phage phiNM1 and constructed strain RNK. V.A.F. and X.D. participated in the conception of the project.

Corresponding authors

Correspondence to David Bikard or Luciano A Marraffini.

Ethics declarations

Competing interests

A patent application (US 61/761,971, PCT/US2014/015252) has been filed related to this work. D.B., L.A.M. and X.D. hold shares in PhageX, a company pursuing applications of this technology.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Tables 1–3 (PDF 7494 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Bikard, D., Euler, C., Jiang, W. et al. Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials. Nat Biotechnol 32, 1146–1150 (2014). https://doi.org/10.1038/nbt.3043

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.3043

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

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