title: 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.
authors.affiliation: 1] MIT Microbiology Program, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. [2] MIT Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
authors.forename: Robert J
authors.identifier: 0000000263975417
authors.initials: RJ
authors.lastname: Citorik
authors.affiliation: 1] MIT Microbiology Program, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. [2] MIT Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Mimee
authors.affiliation: 1] MIT Microbiology Program, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. [2] MIT Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. [3] Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. [4] Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. [5] Harvard Biophysics Program, Harvard University, Boston, Massachusetts, USA. [6] Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.
authors.forename: Timothy K
authors.initials: TK
authors.lastname: Lu
mesh_terms: D000900:Anti-Bacterial Agents / Q000627:therapeutic use; D000890:Anti-Infective Agents / Q000627:therapeutic use; D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence / Q000235:genetics; D064113:CRISPR-Cas Systems; D015780:Carbapenems / Q000627:therapeutic use; D004352:Drug Resistance, Microbial / Q000235:genetics*; D054324:Enterohemorrhagic Escherichia coli / Q000235:genetics* / Q000472:pathogenicity; D018390:Gene Targeting; D010957:Plasmids; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics*; D012260:Ribonucleases / Q000235:genetics*
chemical_list: D000900:Anti-Bacterial Agents; D000890:Anti-Infective Agents; D015780:Carbapenems; D000094704:RNA, Guide, CRISPR-Cas Systems; D012260:Ribonucleases