Rebooting Synthetic Phage-Inducible Chromosomal Islands: One Method to Forge Them All
Synthetic Gram-negative PICI used as targeted therapy. (a) The synthetic Gram-negative PICI exploits the packaging machinery of its temperate phage to pack and deliver a CRISPR-Cas9 (black arrow) cargo into other bacteria. After induction with MC and activation of the ERP cycle, both phage (light green) and PICI (grey) excise from the bacterial (orange) chromosome to be packed into viral particles. Only the PICI-DNA is packed using the cos signaling (red block) to fulfil the viral particles hijacking the TerL of the phage with its cognate Rpp, while the phage-DNA packaging is disabled due to deletion of the cosN site (black cross). Then, the cell is lysed and a lysate containing PICI-only particles is generated. These viral particles containing the synthetic PICI with CRISPR-Cas9 cargo can be delivered as an antimicrobial element, to target and cure plasmids (blue) carrying virulence or AMR genes (purple and yellow arrows). (b) The synthetic EcCICFT073 c1498-c1501::cas9 elements were designed by PCR fragments incorporating the CRISPR-Cas9 system in the adaptable module of the cos island. (c) Deployment and activity of Synthetic PICI with CRISPR-cas9 system against plasmid pRIC carrying the ndm-1 gene. Graphs represent the means of transduced cells with the chloramphenicol resistance cassette (cat) on tetracycline resistant cells (tetR) to measure the proportion of cells cured of pRIC1 with the target gene by transduction of the PICI. Statistical analysis was performed using one-way ANOVA followed by Tuckey’s multiple comparisons test (). Adjusted values for pRIC cas9-∅ versus pRIC cas9-ndm1 , pRIC cas9-∅ versus pRIC-ndm1 cas9-∅, , pRIC cas9-∅ versus pRIC-NDM1 cas9-ndm1 , and pRIC cas9-ndm1 versus pRIC-ndm1 cas9-ndm1 .