. Results .
1. Purification and In Vitro Activity Verification of Cas9 RNP in EcN
a. Lane M: three-color prestained protein Marker;
Lane 1: Bacteriostatic precipitation;
Lane 2: supenatarnt with cleaved bacteria;
Lane 3: Flow-through eluent after binding to nickel beads;
Lanes 4-7: Gradient eluents of different concentrations of imidazole;
b. Lane M: DNA Marker;
Lane 1: target plasmid pCDNA3.1-Flag-PRDX4;
Lane 2: pCold-Cl7-Cas9 -P4 experimental group;
Lane 3: SPEL single digestion;
As shown in Figure 1, the purified RNPs have friendly cleavage activity against the target plasmid.
2. Im7 Protein Display on the Surface of EcN-Engineered Bacteria
This experiment aimed to display the Im7 protein on the surface of EcN cells using the affinity protein InaK (Fig.2). First, the Escherichia coli gene-editing plasmid system pKD46 was used to integrate the T7 RNA polymerase gene, controlled by the lac-uv5 promoter, into the attB site of the EcN genome. As a result, an EcN strain compatible with the pET expression system was successfully engineered. Next, the cryoprotein InaK was fused with the IM7 protein to construct a fusion protein expression plasmid, which was then introduced into the engineered EcN strain. IPTG was added to induce protein expression, and successful production of the InaK-Im7 fusion protein was confirmed by SDS-PAGE electrophoresis (Fig.2).
Lanes 1, 3, 5: Pre-induction; Lanes 2, 4, 6: Post-induction with IPTG.
3. Testing OMV Diameters After Extrusion
To determine whether vesicles of the desired size could be obtained through physical extrusion, we characterized the particle sizes using transmission electron microscopy (TEM) and dynamic light scattering (DLS).
The OMVs containing Cas9 RNP, obtained through EcN self-assembly (Fig.3), were successfully characterized with a particle size of approximately 100 nm.
4. Validation of the Dual-Plasmid Co-Expression Module
The expression levels of Cas9 RNP and Im7 proteins were analyzed by SDS-PAGE and western blotting.
The results showed that the Cas9 RNP and Im7 proteins were successfully expressed by this double plasmid expression system (Fig.4) and that the amount of plasmid transfection was optimal at approximately Cas9 RNP:IM7=1:10.
5. Verification of Anchoring Effect After Extrusion
To assess the impact of physical extrusion on the anchoring of proteins, we validated the system using Dil and sfGFP staining, followed by laser confocal microscopy.
Due to limitations in the resolution of the equipment, we characterized vesicles extruded at 800 nm. Approximately 40% of the vesicles showed successful attachment of elements within the same field of view. We predict that vesicles extruded at 100 nm would exhibit similar ligation efficiency.
6. Verification of OMV Targeting Efficiency
The primary component of OMVs is a phospholipid bilayer membrane, which allows for membrane fusion with mammalian cells, facilitating intracellular delivery. To verify the efficiency of OMVs in delivering target proteins, OMVs containing CL7-mCherry red fluorescent protein were mixed with Im7-attached OMVs in EP tubes for 15 minutes. The mixture was then transferred to HeLa cells for incubation. After approximately 4 hours, fluorescence microscopy at an excitation wavelength of 534 nm was used to visualize the results.
7. Testing Cas9 RNP Gene Editing Efficiency
OMVs containing Cas9 RNPs were delivered into HeLa cells to assess their gene-editing activity. The editing efficiency of the OMV-Cas9 RNPs was compared with that of a commercial liposome transfection reagent using a T7E1 assay (Fig.6).
The results showed that the Cas9 RNP-loaded OMVs exhibited similar gene-editing efficiency to the commercial liposome transfection reagent, with both achieving approximately 40% cleavage of the target gene.
