Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 32 (11), 1141-5

Sequence-specific Antimicrobials Using Efficiently Delivered RNA-guided Nucleases


Sequence-specific Antimicrobials Using Efficiently Delivered RNA-guided Nucleases

Robert J Citorik et al. Nat Biotechnol.


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.

Conflict of interest statement


The authors declare competing financial interests: details are available in the online version of the paper.


Figure 1
Figure 1
RGN constructs delivered by bacteriophage particles (ΦRGN) exhibit efficient and specific antimicrobial effects against strains harboring plasmid or chromosomal target sequences. (a) Bacteriophage-delivered RGN constructs differentially affect host cell physiology in a sequence-dependent manner. If the target sequence is: (i) absent, the RGN exerts no effect; (ii) chromosomal, RGN activity is cytotoxic; (iii) episomal, the RGN leads to either (iiia) cell death or (iiib) plasmid loss, depending on the presence or absence of toxin-antitoxin (TA) systems, respectively. (b) Treatment of EMG2 wild-type (WT) or EMG2 containing native resistance plasmids, pNDM-1 (encoding blaNDM-1) or pSHV-18 (encoding blaSHV-18), with SM buffer, ΦRGNndm-1, ΦRGNshv-18, or multiplexed ΦRGNndm-1/shv-18 at a multiplicity of infection (MOI) ~20 showed sequence-dependent cytotoxicity as evidenced by a strain-specific reduction in viable cell counts (n = 3). CFU, colony-forming units. (c) E. coli EMG2 WT or EMG2 gyrAD87G populations were treated with SM buffer, ΦRGNndm-1 or ΦRGNgyrAD87G at MOI ~20, and viable cells were determined by plating onto Luria-Bertani agar (n = 3).
Figure 2
Figure 2
Characterization of ΦRGN-mediated killing of antibiotic-resistant bacteria. (a) Time-course treatment of EMG2 WT or EMG2 pNDM-1 with SM buffer, ΦRGNndm-1 or ΦRGNshv-18 at a multiplicity of infection (MOI) ~20. Data represent the fold change in viable colonies at indicated time points relative to time 0 h. (b) Dose-response curve of EMG2 WT and EMG2 gyrAD87G treated with various concentrations of ΦRGNgyrAD87G for 2 h. Data represent fold change in viable colonies relative to samples treated with SM buffer. Error bars (a, b), s.e.m. of three independent biological replicates (n = 3). (c) EMG2 E. coli containing the natural pNDM-1 plasmid or the blaNDM-1 gene in a synthetic expression vector (pZA-ndm1-gfp) were treated with either ΦRGNndm-1 or ΦRGNshv-18 at MOI ~ 20 and plated onto both nonselective LB and LB + carbenicillin (Cb) to select for blaNDM-1-containing cells. ΦRGNndm-1 treatment of cells harboring pNDM-1 resulted in a reduction in viability in the absence of selection, whereas ΦRGNndm-1 treatment of cells with pZA-ndm1-gfp demonstrated similar cytotoxicity only under selective pressure for maintenance of the pZA-ndm1-gfp plasmid. (d) EMG2 pSHV-18 complemented with the cognate antitoxin (pZA31-pemI) for the PemK toxin or a control vector (pZA31-gfp) was treated with SM buffer, ΦRGNndm-1 or ΦRGNshv-18. Cultures were plated on LB and LB + Cb and colonies were enumerated to assess cytotoxicity or plasmid loss.
Figure 3
Figure 3
ΦRGN particles elicit sequence-specific toxicity against enterohemorrhagic E. coli in vitro and in vivo. (a) E. coli EMG2 wild-type (WT) cells or ATCC 43888 F′ (EHEC) cells were treated with SM buffer, ΦRGNndm-1 or ΦRGNeae at a multiplicity of infection (MOI) ~100 and plated onto LB agar to enumerate total cell number or LB+kanamycin (Km) to select for transductants with ΦRGNs (n = 3). (b) G. mellonella larvae were injected with either PBS or approximately 4 × 105 colony forming units (CFU) of EHEC. Subsequent administration of ΦRGNeae at MOI ~30 significantly improved survival compared to SM buffer or ΦRGNndm-1 treatment (Log-rank test, P < 0.001). Survival curves represent an aggregate of four independent experiments, each with 20 worms per treatment group (n = 80).
Figure 4
Figure 4
Programmable remodeling of a synthetic microbial consortium. A synthetic population composed of three different E. coli strains was treated with either SM buffer, ΦRGNndm-1, or ΦRGNgyrAD87G at an MOI ~100 and plated onto LB with chloramphenicol, streptomycin or ofloxacin to enumerate viable cells of E. coli CJ236, EMG2 pNDM-1 or RFS289 strains, respectively. ΦRGNndm-1 targets blaNDM-1 in EMG2 pNDM-1 and ΦRGNgyrAD87G targets the gyrAD87G allele in RFS289. Circle area is proportional to total population size and numbers represent viable cell concentrations (CFU/ml) of each strain after the indicated treatment. The s.e.m. based on three independent experiments is indicated in parentheses (n = 3).

Comment in

  • A CRISPR Design for Next-Generation Antimicrobials
    CL Beisel et al. Genome Biol 15 (11), 516. PMID 25417800.
    Two recent publications have demonstrated how delivering CRISPR nucleases provides a promising solution to the growing problem of bacterial antibiotic resistance.

Similar articles

See all similar articles

Cited by 123 PubMed Central articles

See all "Cited by" articles

Publication types

MeSH terms