P1 Bacteriophage-Enabled Delivery of CRISPR-Cas9 Antimicrobial Activity Against Shigella flexneri .

The discovery of clustered, regularly interspaced, short palindromic repeats (CRISPR) and the Cas9 RNA-guided nuclease provides unprecedented opportunities to selectively kill specific populations or species of bacteria. However, the use of CRISPR-Cas9 to clear bacterial infections is hampered by the inefficient delivery of 9 genetic constructs into bacterial cells. Here, we use a broad-host-range P1-derived phagemid to deliver the CRISPR-Cas9 chromosomal-targeting system into and the dysentery-causing to achieve DNA sequence-specific killing of targeted bacterial cells. We show that genetic modification of the helper P1 phage DNA packaging site () significantly enhances the purity of packaged phagemid and improves the Cas9-mediated killing of cells. We further demonstrate that P1 phage particles can deliver chromosomal-targeting phagemids into using a zebrafish larvae infection model, where they significantly reduce the bacterial load and promote host survival. Our study highlights the potential of combining P1 bacteriophage-based delivery with the CRISPR chromosomal-targeting system to achieve DNA sequence-specific cell lethality and efficient clearance of bacterial infection.