P1 Bacteriophage-Enabled Delivery of CRISPR-Cas9 Antimicrobial Activity Against <i>Shigella flexneri</i>

Abstract

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 <i>in vivo</i> is hampered by the inefficient delivery of <i>cas</i>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 <i>Escherichia coli</i> and the dysentery-causing <i>Shigella flexneri</i> to achieve DNA sequence-specific killing of targeted bacterial cells. We show that genetic modification of the helper P1 phage DNA packaging site (<i>pac</i>) significantly enhances the purity of packaged phagemid and improves the Cas9-mediated killing of <i>S. flexneri</i> cells. We further demonstrate that P1 phage particles can deliver chromosomal-targeting <i>cas9</i> phagemids into <i>S. flexneri</i> <i>in vivo</i> 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.