. Design .

1. Overall experimental design

Module 1: High-Efficiency In Vivo Assembly of Cas9 RNP

This module focuses on the efficient in vivo self-assembly of Cas9 ribonucleoproteins (RNPs). Building on the existing technologies in our laboratory, we aim to enhance the expression levels of guide RNA (gRNA) and improve the assembly efficiency of Cas9 RNPs. To achieve this, we replaced the Tac promoter upstream of the gRNA with a strong T7 promoter and introduced the T7 RNA polymerase gene into the EcN genome via a knock-in approach. Following this, we will purify the Cas9 RNP from EcN and conduct in vitro activity assays to validate its function.

Module 2: Design of Targeting Elements

This module involves designing two distinct targeting element strategies, referred to as Plan A and Plan B. Additionally, we will verify the surface display of these targeting elements on engineered EcN bacteria.

Plan A: CL7 is a modified version of the DNase domain of Colicin E7, retaining its strong affinity for its natural inhibitor protein, Im7. We aim to display Im7 on the bacterial membrane by linking it to the ice-nucleation protein (InaK). By fusing single-chain antibodies or nanobody complexes, each possessing different targeting properties, with the CL7 tag, we seek to achieve targeted delivery to specific cell types.

Plan B: Recent studies have developed structures capable of binding to the fragment crystallizable (Fc) region of antibodies. From these studies, the z and 4z domains, which exhibit high affinity for the Fc domain, have been identified. We will link these domains to the InaK protein and display them on the outer membrane of EcN. Commercially available monoclonal antibodies with Fc domains will be utilized for targeted delivery to specific cells.

Module 3: Dual-Plasmid Co-expression System

In this module, we will optimize the expression levels of Cas9 RNP and Im7 proteins through SDS-PAGE and Western blot analysis. The optimal transfection ratios for the required plasmids will also be determined.

Module 4: Physical Extrusion

In this module, the design draws on the "LUVET" technology. The engineered bacteria EcN after protoplast formation is repeatedly extruded under moderate pressure through a polycarbonate filter membrane, and polycarbonate filter membranes of different sizes are used to obtain artificial outer - membrane vesicles (OMVs) with the target particle size. After extrusion, we will characterize the size and morphology of the artificial OMVs and test the anchoring effect of parts.

Module 5: Validation of OMV-Mediated Target Cell Delivery

OMVs are composed of a phospholipid bilayer, which can fuse with mammalian cells via membrane fusion, allowing for the delivery of encapsulated materials into the target cells. In this module, we will assess the capacity of OMVs to deliver Cas9 RNP through cellular-level experiments.

Module 6: Evaluation of Gene-Editing Efficiency

In this module, we will compare the gene-editing efficacy of the engineered OMV-Cas9 RNP with that of commercial liposome-based transfection reagents. This will allow us to estimate the relative efficiency of gene editing in target cells.