(146b) Crispr-Based Antimicrobials for Treating Multidrug-Resistant Infections

Once considered a revolution in modern medicine, antibiotics are becoming obsolete. Multidrug-resistant infections are increasingly being reported, where many resistance mechanisms can be readily passed between unrelated bacteria. Furthermore, traditional antibiotics are broad-spectrum, decimating the multitude of beneficial bacteria in the human body and leaving us susceptible to opportunistic infections and chronic illnesses. These challenges demand novel approaches to treat multidrug-resistant infections while discriminating between pathogenic and symbiotic bacteria.

Here, we report a targeted strategy for treating ranging bacterial infections that relies on CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR associated) systems. These RNA-directed defense systems are naturally employed by bacteria and archaea to recognize and cleave the DNA of foreign invaders. Small CRISPR RNAs bind their complementary sequences in the foreign DNA directing cleavage or degradation by Cas proteins. By designing and expressing synthetic genome-targeting CRISPR RNAs, we can reprogram CRISPR-Cas systems to induce bacterial cell death. We found that this strategy could achieve potent and sequence-specific killing of multidrug-resistant bacteria regardless of the type or location of the targeted region and using either imported or native CRISPR-Cas systems. Additionally, we were able to distinguish between even highly related strains as well as between commensal and pathogenic bacteria.

For this strategy to further develop as a therapeutic, a delivery method for the CRISPR-Cas DNA is needed. Toward this goal, we are engineering the broad-host range P1 bacteriophage as a delivery platform that can be used for multiple enteric pathogens. We identified and validated multiple landing sites to introduce foreign DNA while maintaining the bacteriophage function. The engineered P1 bacteriophage showed significant success in delivering the DNA of interest to the target bacteria compared to the state-of-art P1 phagemid systems. Finally, we utilized the same approach to deliver genome-targeting CRISPR-Cas systems to a number of gram-negative pathogens and eradicate them. We envision using this strategy to develop â??smartâ? non-antibiotic treatments capable of circumventing common modes of resistance while readily distinguishing between pathogens and symbiotic bacteria.