RNA-Guided Nucleases As Programmable-Spectrum Antimicrobials and Microbial Population Sculptors

With the rising tide of antibiotic resistance and concomitant dearth of novel antimicrobials, new paradigms to treat bacterial infections are in dire need. Unlike conventional, broad-spectrum antibiotics, which target pathogenic and commensal bacterial populations alike, we developed a programmable-spectrum antimicrobial whose activity is dependent on the presence of genetic signatures in microbial cells. By rewiring the Type II CRISPR-Cas system of Streptococcus pyogenes, we created RNA-guided nucleases (RGNs) that mediate double-stranded breaks in genes or single-nucleotide polymorphisms (SNPs) associated with drug resistance or virulence. Using bacteriophage particles (ΦRGN) or conjugative vectors (mRGN), RGNs could be efficiently delivered to bacterial populations to enact sequence-specific killing. When targeting a chromosomal SNP which confers resistance to quinolone class antibiotics, ΦRGNs were able to elicit a greater than 4-log reduction in viable cells, while having no activity against the wild-type parental strain. In addition to targeting the chromosome, we found that using ΦRGNs to target the bla_NDM-1 and bla_SHV-18 genes on multidrug resistance plasmids caused a 2-3-log reduction through the artificial activation of co-harbored toxin-antitoxin systems. Overexpression of the cognate antitoxin resulted in curing of pSHV-18 and antibiotic resensitization. To further demonstrate specificity of our particles, we treated a consortium of three E. coli strains with ΦRGNs against the unique resistance markers of each strain and observed selective killing of only the desired strain. Finally, we designed constructs against a virulence gene in enterohemorrhagic E. coli O157:H7 and used the ΦRGNs to significantly improve survival in a wax moth larva infection model.