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 Cas91, 2 delivered by a bacteriophage. We show that Cas9 re-programmed to target virulence genes kills virulent, but not avirulent, Staphylococcus aureus. Re-programming the nuclease to target antibiotic resistance genes destroys staphylococcal plasmids that harbor antibiotic resistance genes3, 4 and immunizes avirulent staphylococci to prevent the spread of plasmid-borne resistance genes. We also demonstrate the approach in vivo, showing its efficacy against S. aureus in a mouse skin colonization model. This new technology creates opportunities to manipulate complex bacterial populations in a sequence-specific manner.

Antimicrobial bacteriophage-derived proteins and therapeutic applications

“…Since a large variety of phages exist for any given bacterium, the current trend is the use of 'cocktail' preparations to ensure the presence of multiple phage that target the pathogen in a variety of mechanisms, thus reducing the relatively high frequency of phage resistance exhibited by bacteria. 7,8 Phage therapies are also self-dosing, increasing in numbers at the site of infection only in the presence of their host bacteria. Most phages do not directly display any risk to human health or the environment.…”

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.…”