. Engineering .

Overview

Synthetic biology is an interdisciplinary science that seeks to design and build new biological parts and systems to perform specific functions. To ensure that a product meets final expectations, it needs to go through an engineering cycle of design, build, test, and learn. Our team applied this philosophy to the project by designing a targeted gene-edited delivery system based on the outer membrane vesicles of the probiotic Escherichia coli Nissle 1917 (EcN). By engineering EcN, using "self-assembly" biosynthesis to produce CRISPR/Cas9 ribonucleoprotein complex (RNP), displaying pairing elements or targeting elements on its surface, and then processing the constructed strains into protoplasts.The ideal size of bacterial outer membrane vesicles (OMVs) carrying Cas9 RNP can be obtained by liposome extruder to achieve the targeted delivery of Cas9 RNP, providing new possibilities for gene therapy.

Cycle 1

Self-assembly synthesis of spCas9 RNP in wild-type EcN

Design

The CRISPR-Cas9 system can be delivered into an organism for gene editing in the form of a plasmid vector, mRNA or Cas9 RNP. Cas9 RNP, which has advantages due to its fast editing, low off-target rate and weak immune response, has been used to develop gene therapy tools for genetic diseases. However, the traditional in vitro assembled form of synthetic Cas9 RNP is inefficient, costly, and its activity cannot be maintained stably. Here, we propose a one-step biosynthesis of Cas9 RNP via wild-type EcN using the laboraty-pioneered E. coli expression synthesis method[1] (Fig. 1).

Fig.1 Schematic diagram of Cas9 RNP synthesis process of E. coli by "biological self-assembly"

Build
Based on previous work in the laboratory, we constructed in vivo self-assembled plasmids of Cas9 RNP, in which both the expression of Cas9 protein and the transcription unit of gRNA utilize Tac promotors that can be recognized by E. coli RNA polymerase. The plasmids were transferred into wild-type EcN for expression.

Tests
We conducted protein purification and in vitro activity verification of EcN-expressed Cas9 RNP (Fig. 2) to test its efficiency in cutting target plasmids.

Fig.2 Purification effect and in vitro activity verification of Cas9 RNP

Learning
The experimental results show that the yield of Cas9 RNP synthesized by Tac promoter is about 1 mg per liter of fermentation medium, which is far lower than the experimental expectation. The analysis may be due to the fact that Tac promoter is not a strong promoter, and the transcriptional effect of gRNA is poor, resulting in a low level of fully assembled RNP with enzyme activity.

Cycle 2

Design

We learned that wild EcN does not have a T7 RNA polymerase that can recognize the T7 strong promoter. Therefore, in order to improve the expression of gRNA and the assembly efficiency of Cas9 RNP, we designed an experiment to tap into the T7 RNA polymerase gene in the EcN genome.

Build
By phage homologous recombination, we inserted T7 RNA polymerase gene into EcN genome and reconstructed the expression plasmid of Cas9 RNP, in which the Cas9 protein was expressed by Tac promoter, and the transcription products of gRNA were obtained by T7 activation, and the Cas9 RNP was synthesized by self-assembly in EcN bacteria.

Tests
We utilized SDS-PAGE to compare the expression levels of Cas9 RNP in the newly constructed EcN strain using the T7 promoter with those of the previous cycle using the tac promoter. The results indicated that the protein expression level of the EcN strain with the T7 promoter was significantly higher than that with the tac promoter (Fig.3). Additionally, we successfully validated the gene editing activity of the Cas9 RNP expressed by the newly constructed EcN strain through in vitro cleavage assays (Fig.4).

Fig.3 Results of SDS-PAGE experiment on expression of purified CAS9 RNP

Fig.4  In vitro cleavage of the target plasmid
Lane M: DNA Marker
Lane 1: 5483bp Target Plasmid
Lane 2: Xba I Single-Enzyme Restriction
Lane 3: spCas9 RNP Exogenous Enzyme Cleavage

In addition, OMVs were prepared by liposome extruder, purified and characterized by transmission electron microscopy and dynamic light scattering. The results showed that OMVs containing Cas9 RNP was successfully obtained by EcN self-assembly (Fig. 5), and the particle size was about 100 nM. The expression level of Cas9 RNP reached 8 mg per liter of fermentation medium, which had excellent enzyme digestion activity.

Fig.5 TEM image analysis and DLS analysis results of OMVs

Learning
The results show that EcN constructed by genetic engineering can be used as chassis cells for the subsequent preparation of OMVs delivery vector experiments.

Cycle 3

Design - Path 1

The dissociation constant Kd of the Colicin E7 DNase (CE7) domain with its immune protein 7 (Im7) reaches 10^-14 to 10^-17 M. CL7, an engineered variant of CE7, loses DNase activity but maintains a high affinity with Im7. We designed this pairing element to enable easy access to a variety of targeting plug-ins (Fig.6).

Fig.6 Schematic diagram of the targeting plug-in pairing system for Im7 protein to CL7 fusion pair displayed on the EcN surface.

Firstly, we designed two plasmids. One displayed Im7 protein on the surface of EcN membrane, and Im7 was anchored to the cell surface by the extracellular membrane anchor protein InaK. Secondly, we constructed a CL7-fused fluorescent protein sfGFP to verify the successful display of the foreign target protein on the EcN surface. Among them, we designed 1xlinker and 3xlinker.

Build
We transferred the pCold-Inak-Im7 plasmid into EcN and pET23a-CL7-sfGFP into Escherichia coli BL21. Then, it was verified whether EcN successfully expressed Im7 protein and BL21 successfully expressed CL7-sfGFP. The engineered EcN was prepared into protoplasts, and OMVs with different pore sizes were obtained by liposome extruder.

Tests
We first incubated EcN engineered bacteria with Im7 protein displayed on their surfaces with purified CL7-sfGFP fluorescent protein and washed away the non-specifically bound GFP. Under a fluorescence microscope, the results (Fig.7) showed that Im7 successfully displayed and was able to bind specifically to CL7-fused proteins, and the results of experiments containing 3xlinker were significantly better than those containing 1xlinker.

Fig.7 (a) Bright field of view of negative control;(b) the image of the negative control under 488 nm excitation light;(c)Bright field of view in EcN/pCold-InaK-Im7 (1xlinker) experimental group;(d)Images of EcN/pCold-InaK-Im7 (1xlinker) experimental group under 488 nm excitation light,(e)bright field of EcN/pCold-InaK-Im7 (3xlinker) experimental group;(f)Images of EcN/pCold-InaK-Im7 (3xlinker) experimental group under 488 nm excitation light

Next, we incubated the OMVs obtained from the liposome extruder with purified CL7-sfGFP fluorescent protein, and washed away the non-specifically bound GFP. Observation under a fluorescence microscope showed that the results (Fig.8) were also consistent with expectations, indicating that the prepared OMVs delivery vector was able to bind the CL7 fusion protein.

Fig.8 (a) negative control under 488 nm excitation light, and (b) OMVs/pCold-ClyA-Im7 experimental group under 488 nm excitation light

Design - Path 2

In addition to the above Im7-CL7 based superaffinity pairing system, we also learned through literature review that some bacterial proteins such as Staphylococcus aureus protein A, Streptococcal protein G, etc., can bind[3] specifically to the Fc end of antibodies (such as IgG). Based on this, we designed the second path as follows: express these bacterial proteins on the membrane surface of EcN, and then incubate the OMVs with these proteins displayed on the membrane surface directly with the antibodies with Fc domain purchased from the company to realize the display of antibodies on the surface of OMVs. Therefore, we chose z domain as EcN surface display protein here.

Fig.9 Schematic diagram of the EcN surface displaying the Z domain or 4Zd domian for binding to commercial antibodies

Build
Plasmid construction has been completed for this part of the EcN and expression testing is under way.

Learning
The experimental results show that the EcN outer membrane display system constructed by genetic engineering can meet the expectations, and can display the designed paired binding elements on the surface, and verify the physical extrusion method to obtain OMVs, which displays the targeted element binding plug-in on the surface.

Cycle 4

Design

EcN two-plasmid system for co-expressing Cas9 RNP and surface display elements.

Build
We transferred the expression plasmid of ampicillin resistant Cas9 RNP and the display plasmid of kanamycin resistant Im7 into the engineered EcN strain for expression (Fig.10), and investigated their concentration ratios in order to obtain the best protein expression levels.

Fig.10 Schematic diagram of the double plasmid expression system

Tests
By SDS-PAGE and western-blot, we demonstrated that Cas9 RNP and Im7 proteins could be successfully expressed by this two-plasmid expression system (Fig.11), and the optimal transfection volume of the plasmid was about Cas9 RNP: Im7=1:10.

Fig.11 Cas9 RNP and Im7 proteins were successfully expressed by the dual-plasmid expression system

Cycle 5

Design

Cell genome editing experiment and targeted delivery system test.

Build
First, we delivered OMVs coated with Cas9 RNP into HeLa cells for testing, targeting PRDX4 gene, and verifying Indel gene editing activity; Next, the transmembrane peptide and monoclonal antibody expressed by fusion of CL7 protein were incubated with it to verify its cell targeting and gene editing activity.

Tests
Through the classic T7E1 experiment, we have verified that the prepared OMVs-Cas9 RNP has comparable cellular genome editing activity to the commercial liposomal transfection reagent(Fig.12).

Fig.12 Verification of genome editing activity in T7E1 cells

At present, we are verifying the results of cell gene editing after the insertion of the targeted element.

References:

1. Qiao J, Li W, Lin S, Sun W, Ma L, Liu Y. Co-expression of Cas9 and single-guided RNAs in Escherichia coli streamlines production of Cas9 ribonucleoproteins. Commun Biol. 2019; 2:161.Published 2019 May 3. doi:10.1038/s42003-019-0402-x
2. Zhao, M., Cheng, X., Shao, P. et al. Bacterial protoplast-derived nanovesicles carrying CRISPR-Cas9 tools re-educate tumor-associated macrophages For enhanced cancer immunotherapy. Nat Commun 15, 950 (2024).
3. Wiklander, O.P.B., Mamand, D.R., Mohammad, D.K. et al. Antibody-displaying extracellular vesicles for targeted cancer therapy. Nat. Biomed. Eng (2024).