Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials

Journal name:
Nature Biotechnology
Volume:
32,
Pages:
1146–1150
Year published:
DOI:
doi:10.1038/nbt.3043
Received
Accepted
Published online

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.

At a glance

Figures

  1. Sequence-specific killing of S. aureus by a phagemid-delivered CRISPR system.
    Figure 1: Sequence-specific killing of S. aureus by a phagemid-delivered CRISPR system.

    (a) The ΦNM1 phage delivers the pDB121 phagemid to S. aureus cells. pDB121 carries the S. pyogenes tracrRNA, cas9 and a programmable CRISPR array sequence. Expression of cas9 and a self-targeting crRNA leads to chromosome cleavage and cell death. (b) Lysates of pDB121 phagemid targeting the aph-3 kanamycin resistance gene or a nontargeting control are spotted on top-agar lawns of either RNΦ or RNKΦ cells. Scale bar, 5 mm. (c) Treatment of RNΦ (blue triangles) or RNKΦ (red diamonds) with pDB121double colonaph at various MOI. Survival is calculated as the ratio of CFU recovered after treatment to CFU from an untreated sample of the same culture (mean ± s.d.). The black curve represents the probability that a cell does not receive a phagemid making the assumption that all cells have the same chance of receiving phagemid. (d) Time course of treatment of RNKΦ/pCN57 (GFP reporter plasmid) cells in a mixed culture with nontargeted RNΦ cells. Plain lines show OD and dashed lines GFP (mean ± s.d.). Kanamycin (25 μg/ml) is shown in orange, streptomycin (10 μg/ml) in green, pDB121double colonaph (MOI ~20) in purple, pDB121double colonØ (MOI ~20) in blue.

  2. Targeting antibiotic resistance genes and plasmids in an MRSA strain.
    Figure 2: Targeting antibiotic resistance genes and plasmids in an MRSA strain.

    (a) Treatment of a mixed population of RNΦ and USA300Φ results in killing of the targeted USA300 MRSA strain and delivery of an immunizing phagemid to the rest of the population. (b) pDB121double colonmecA specifically kills USA300Φ in a mixed population. Exponentially growing USA300Φ and RNΦ cells were mixed 1:1 and treated with pDB121 at an MOI of ~5. Cells were plated either on a nonselective medium, on chloramphenicol-containing medium to measure the proportion of cells receiving the phagemid treatment, or on oxacillin-containing medium to measure the proportion of USA300Φ cells in the population (mean ± s.d.). (c) The CRISPR array sequence is programmed to target the pUSA01 and pUSA02 plasmids simultaneously. (d) USA300Φ was treated with pDB121 lysates targeting each plasmid individually or in combination. Cells were plated either on a nonselective medium, on chloramphenicol-containing medium to measure the proportion of cells receiving the phagemid treatment, or on tetracycline-containing medium to measure the proportion of cells cured of pUSA02 (mean ± s.d.). (e) Plasmid curing was confirmed by the lack of PCR amplification with plasmid-specific oligonucleotides in eight independent CFUs after treatment with the double targeting construct. (f) A population of RNΦ cells was immunized against plasmid horizontal transfer by treatment with the pUSA02-targeting pDB121 phagemid. 30 min after treatment, the population was transduced with a ΦNM1 stock grown on USA300. Cells were plated either without selection or on tetracycline to measure transduction efficiency of the pUSA02 plasmid (mean ± s.d.).

  3. Sequence-specific killing of kanamycin-resistant S. aureus in a mouse skin colonization model.
    Figure 3: Sequence-specific killing of kanamycin-resistant S. aureus in a mouse skin colonization model.

    Mice skin was colonized with a 1:1 mixture of 105 RNΦ and RNKΦ cells carrying the pCN57 GFP reporter plasmid, followed by treatment at 1 h with PBS (n = 15), ΦNM1 (n = 16), pDB121double colonaph (n = 14) or mupirocin (n = 10). After 24 h the skin from the treated area was excised, homogenized and serial dilutions of the homogenate was plated on mannitol salt agar. (a) Pictures of two representative plates exposed to a wavelength enabling visualization of GFP. Scale bar, 1 cm. (b) The proportion of RNKΦ cells in the population was measured as the proportion of green fluorescent CFU on the plates. Data points indicate individual mice; black lines represent the mean ± s.d.

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Author information

  1. These authors contributed equally to this work.

    • Chad W Euler &
    • Wenyan Jiang

Affiliations

  1. Laboratory of Bacteriology, The Rockefeller University, New York, New York, USA.

    • David Bikard,
    • Wenyan Jiang,
    • Philip M Nussenzweig,
    • Gregory W Goldberg &
    • Luciano A Marraffini
  2. Laboratory of Bacterial Pathogenesis and Immunology, The Rockefeller University, New York, New York, USA.

    • Chad W Euler &
    • Vincent A Fischetti
  3. INRIA Paris-Rocquencourt, Rocquencourt, France.

    • Xavier Duportet
  4. Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Xavier Duportet
  5. Present address: Synthetic Biology Group, Institut Pasteur, Paris, France.

    • David Bikard

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.

Competing financial 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.

Corresponding authors

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