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Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases

Abstract

Current antibiotics tend to be broad spectrum, leading to indiscriminate killing of commensal bacteria and accelerated evolution of drug resistance. Here, we use CRISPR-Cas technology to create antimicrobials whose spectrum of activity is chosen by design. RNA-guided nucleases (RGNs) targeting specific DNA sequences are delivered efficiently to microbial populations using bacteriophage or bacteria carrying plasmids transmissible by conjugation. The DNA targets of RGNs can be undesirable genes or polymorphisms, including antibiotic resistance and virulence determinants in carbapenem-resistant Enterobacteriaceae and enterohemorrhagic Escherichia coli. Delivery of RGNs significantly improves survival in a Galleria mellonella infection model. We also show that RGNs enable modulation of complex bacterial populations by selective knockdown of targeted strains based on genetic signatures. RGNs constitute a class of highly discriminatory, customizable antimicrobials that enact selective pressure at the DNA level to reduce the prevalence of undesired genes, minimize off-target effects and enable programmable remodeling of microbiota.

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Figure 1: RGN constructs delivered by bacteriophage particles (ΦRGN) exhibit efficient and specific antimicrobial effects against strains harboring plasmid or chromosomal target sequences.
Figure 2: Characterization of ΦRGN-mediated killing of antibiotic-resistant bacteria.
Figure 3: ΦRGN particles elicit sequence-specific toxicity against enterohemorrhagic E. coli in vitro and in vivo.
Figure 4: Programmable remodeling of a synthetic microbial consortium.

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Acknowledgements

We would like to thank R. Meyer (Institute for Cell and Molecular Biology, University of Texas Austin) for R1162, D.L. Court (Center for Cancer Research, National Cancer Institute at Frederick) for pSIM9, and A.R.M. Bradbury (Los Alamos National Laboratory) for M13cp. The authors thank J. Rubens for assistance with flow cytometry experiments and H. Gancz and D. Zurawski for assistance with the Galleria infection model. T.K.L. acknowledges support from the US National Institutes of Health (NIH) New Innovator Award (1DP2OD008435), an NIH National Centers for Systems Biology grant (1P50GM098792), the Defense Threat Reduction Agency (HDTRA1-14-1-0007), the US Army Research Laboratory and the US Army Research Office through the Institute for Soldier Nanotechnologies (W911NF13D0001) and the Henry L. and Grace Doherty Professorship in Ocean Utilization. R.J.C. is supported by funding from the NIH/National Institute of General Medical Sciences Interdepartmental Biotechnology Training Program (5T32 GM008334), and M.M. is a Howard Hughes Medical Institute International Student Research fellow and a recipient of a Fonds de recherche Santé Québec Master's Training Award.

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R.J.C. and M.M. designed and performed experiments. R.J.C., M.M. and T.K.L. conceived this study, analyzed the data, discussed results and wrote the manuscript.

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Correspondence to Timothy K Lu.

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R.J.C., M.M. and T.K.L. have filed a provisional application with the US Patent and Trademark Office on this work.

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Citorik, R., Mimee, M. & Lu, T. Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases. Nat Biotechnol 32, 1141–1145 (2014). https://doi.org/10.1038/nbt.3011

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