BioDesign Research / 2020 / Article / Fig 3

Research Article

Rebooting Synthetic Phage-Inducible Chromosomal Islands: One Method to Forge Them All

Figure 3

Engineered SaPIs with CRISPR-Cas9 for targeted therapy. (a) The synthetic SaPI 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 SaPI (grey) excise from the bacterial (red) chromosome to be packed into viral particles. Only the SaPI-DNA is packed with its cognate TerS (green arrow) and the TerL of the phage, while the phage-DNA packaging is disabled due to the deletion of the phage TerS (black cross). Then, the cell is lysed and a lysate containing SaPI-only particles is generated. These viral particles containing the synthetic SaPI with CRISPR-Cas9 cargo can be then deployed as antimicrobial elements to target and eliminate bacteria with the mecA sequence (yellow arrow). (b) All of the synthetic SaPIbov2 variants except the synthetic island with CRISPR-Cas9 targeting rsaE showed levels of transduction equal to the parent version induced under a background of packaging defective 80α ΔterS. Graphs represent transduction titers performed in RN4220. Statistical analysis was performed using two-way ANOVA followed by Dunnett’s multiple comparisons test using the parent SaPIbov2 bap::tetM as control and as reference for all comparisons (). Adjusted values for rebooted SaPIbov2 bap::tetM , SaPIbov2::tetM-cas9-∅ , SaPIbov2::tetM-cas9-mecA , SaPIbov2::Pcad-cas9--tetM , and SaPIbov2::Pcad-cas9-rsaE-tetM . (c) A SaPIbov2 engineered with cadmium-inducible CRISPR-Cas9 system targeting rsaE was used to kill S. aureus. RN4220. RN4220 ΔrsaE strain was used as control to show specific killing by target. Cadmium concentrations were used at 1 μM. (d) Graphs represent transduction titers performed in RN4220. Statistical analysis was performed using two-way ANOVA followed by Dunnett’s multiple comparisons test using infection with SaPIbov2 bap::tetM as control for each strain and as reference for all comparisons (). Adjusted values for infection of RN4220 ΔrsaE with SaPIbov2::Pcad-cas9--tetM , and SaPIbov2::Pcad-cas9-rsaE-tetM ; and for infection of RN4220 with SaPIbov2::Pcad-cas9--tetM , and SaPIbov2::Pcad-cas9-rsaE-tetM . (e) Killing of methicillin-resistant S. aureus USA100 and USA200 was performed by using synthetic SaPIbov2::tetM-cas9 containing CRISPR-Cas9 targeting mecA. A SaPIbov2::tetM-cas9 version with null (∅) gRNA was used as control. (f) Graphs represent transduction titers performed in MRSA strains. Statistical analysis was performed using two-way ANOVA followed by Dunnett’s multiple comparisons test using infection with SaPIbov2 bap::tetM as control for each strain and as reference for all comparisons (). Adjusted values for infection of RN4220 with SaPIbov2::tetM-cas9-∅ , and SaPIbov2::tetM-cas9-mecA ; for infection of USA100 with SaPIbov2::tetM-cas9-∅ , and SaPIbov2::tetM-cas9-mecA ; for infection of USA200 with SaPIbov2::tetM-cas9-∅ , and SaPIbov2::tetM-cas9-mecA .