Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials

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 genes 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.

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 non-targeting control are spotted on top-agar lawns of either RN^Φ or RNK^Φ cells. Scale bar, 5mm (c) Treatment of RN^Φ (blue triangles) or RNK^Φ (red diamonds) with pDB121::aph 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 RNK^Φ/pCN57 (GFP reporter plasmid) cells in a mixed culture with non-targeted RN^Φ cells. Plain lines show OD and dashed lines GFP. Kanamycin (25 ug/ml) is shown in orange, streptomycin (10 ug/ml) in green, pDB121::aph (MOI ~20) in purple, pDB121::Ø (MOI ~20) in blue.

Figure 2

Targeting antibiotic resistance genes and plasmids in an MRSA strain. (a) Treatment of a mixed population of RN^Φ and USA300^Φ results in killing of the targeted USA300 MRSA strain and delivery of an immunizing phagemid to the rest of the population. (b) pDB121::mecA specifically kills USA300^Φ in a mixed population. Exponentially growing USA300^Φ and RN^Φ cells were mixed 1:1 and treated with pDB121 at an MOI of ~5. Cells were plated either on a non-selective 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 USA300^Φ cells in the population (mean ± s.d.). (c) The CRISPR array sequence is programmed to target the pUSA01 and pUSA02 plasmids simultaneously. (d) USA300^Φ was treated with pDB121 lysates targeting each plasmid individually or in combination. Cells were plated either on a non-selective 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 8 independent CFUs after treatment with the double targeting construct. (f) A population of RN^Φ cells was immunized against plasmid horizontal transfer by treatment with the pUSA02-targeting pDB121 phagemid. 30 min after treatment, the population is transduced with a ΦNM1 stock grown on USA300. Cells are plated either without selection or on tetracycline to measure transduction efficiency of the pUSA02 plasmid (mean ± s.d.).

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 RN^Φ and RNK^Φ cells carrying the pCN57 GFP reporter plasmid, followed by treatment at 1 hour with phosphate buffer saline (n=15), ΦNM1 (n=16), pDB121::aph (n=14) or mupirocin (n=10). After 24 hours 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 RNK^Φ 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 ± SD.