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.

Temperate and lytic bacteriophages programmed to sensitize and kill antibiotic-resistant bacteria

“…The effectiveness of the CRISPR-Cas system in eliminating plasmid DNA and lytic phages is well established (1,2,15,19,26). Nevertheless, its utility in clinical settings as a tool to render pathogens sensitive to antibiotics and to reduce horizontal gene transfer of resistance determinants has only recently been demonstrated (16,17). We believe that the strategy provided here can be applied to different pathogen-phage systems as phages can be found for most of the pathogens, and a compatible CRISPR-Cas system should work in many pathogens.…”

“…Two recent elegant studies demonstrated that phage-transferable CRISPR-Cas systems are capable of specifically killing pathogens or resensitizing them to antibiotics (16,17). These studies, and another study (13), also showed that the transferred CRISPR-Cas system is capable of eliminating specific bacterial populations.…”

“…CRISPR-Cas systems have also recently been used to phenotypically correct genetic diseases in live animals (11), and their utility is being explored for various therapeutic approaches in mammals. Nevertheless, only limited studies have shown the use of CRISPR-Cas systems to target antibiotic resistance genes or a specific population of virulent bacterial strains (12)(13)(14)(15)(16)(17).…”

Programmable sensitivity

“…Two independent teams believe they could have a farreaching solution in the gene editing technology CRISPR, which allows them to tailor therapies to specific resistance mutations. 1,2 The most promising application of the findings is the potential to resensitize resistant bacteria to standard antibiotics, but the researchers will have Figure 1. CRISPR's double-edged knife.…”

Could CRISPR be the solution for gene editing’s Gordian knot?

“…Cas9 can change the sensitivity and responses of cells to discovered drug, evidenced by the finding that CRISPR/Cas RGNs could alter the resistance of multidrugs in the model of a Galleria mellonella infection (Citorik et al 2014). The preliminary findings from Church and his colleagues showed that Cas9 could inactivate 62 porcine endogenous retroviruses (PERVs) in pig embryos (Nature News 2016).…”