Engineered Broad Host Range Phage for Delivery of CRISPR Antimicrobials

Collias, D., North Carolina State University
Fagen, J., North Carolina State University
Beisel, C. L., North Carolina State University
DNA cleavage by RNA-guided CRISPR nucleases has been shown to be lethal in bacteria [1,2,3] and has given rise to the development of CRISPR antimicrobials. This technology is programmable by modifying the guide to target specific strains of bacteria [1,2,3]. This comes at an opportune time as antibiotic resistance in bacteria continues to increase. Since implementing CRISPR-Cas systems towards antimicrobial applications, the next challenge was broadly delivering these systems in diverse bacterial communities. We developed an engineered, broad host range P1 bacteriophage as a CRISPR antimicrobial delivery vehicle towards various strains of gammaproteobacteria. We were interested in determining if P1 would be able to package its genome while also encoding for a CRISPR nuclease and guide. As a result, the delivery efficiency based on packaged DNA lengths was determined and we were able to confirm that P1 can package and deliver its genome and DNA encoding for a CRISPR nuclease. Furthermore, we identified locations on the P1 genome to incorporate DNA without impacting particle production and subsequent infection into hosts of interest. Finally, we compared production and infection efficiencies of P1 encoding for Cas9 and Cas12a as a head-to-head comparison of single effectors. CRISPR-Cas systems have been demonstrated as a highly specific and programmable antimicrobial technology that is currently limited by the ability to engineer a broad delivery vehicle.


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