Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials

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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.

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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.

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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

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.

Correspondence to David Bikard or Luciano A Marraffini.

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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.

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

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

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