. Description .

1. Inspiration

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2. Background

Off-Target effect of gene editing technology

2.1 Gene editing technology

Gene editing technology, especially the CRISPR/Cas9 system, has been widely used in life science research and clinical treatment due to its simple and efficient characteristics. However, the off-target effect is one of the main challenges facing this technology. Off-target effects refer to gene editing tools accidentally cutting DNA in non-target regions, resulting in unintended gene mutations. This effect may have adverse effects on organisms, including cellular dysfunction, disease development, etc.

2.2 There are two main reasons for the off-target effect:

1. The problem of the tool itself: the sgRNA (single guide RNA) in the CRISPR/Cas9 system is responsible for guiding the Cas9 protein to a specific gene sequence. Different sgRNA lengths and sequences affect the ability of Cas9 and thus the possibility of off-target.
2. External influences: The application of gene editing technology usually needs to be carried out in cells or in vivo. Positions on the nucleus or chromosomes, different cell states and overall factors may affect the accuracy and stability of Cas9 and sgRNA complexes, leading to off-target effects occur. The occurrence of off-target effect may have a negative impact on the application of gene editing technology, and even lead to experimental failure. For example, for the treatment of stem cells or embryo gene editing, off-target effects may induce malignant changes, tumors and other safety risks.

2.3 For the off-target effect, researchers have resorted to a variety of strategies:

1. Tools for predicting off-target effects: Potential off-target sites are predicted through software, and then aligned through genome sequencing to confirm whether there is off-target.
2. New gene editing systems: For example, the new CRISPR/Cas system has higher accuracy than the CRISPR/Cas9 system and can have significant off-target effects.
3. Design targets with high reference genomes: ensure the accuracy of gene editing and avoid off-target due to errors in the reference genome.
4. Optimize the delivery system of gene editing tools: somatic clone mutations generated in tissue culture, off-target or improve the accuracy of gene editing.
Although the off-target effect of gene editing technology is still a challenge, with the deepening of research and the continuous advancement of technology, the rapid development of gene editing technology has provided new possibilities for medical research and treatment. However, the existence of off-target effects limits the wide application of gene editing technology. Therefore, researchers are working to understand the mechanism of off-target effects, to develop new off-target effects, and to improve the delivery system of gene editing tools.

3. The problem

The safe and efficient delivery of CRISPR-Cas9

The delivery mode of CRISPR / Cas 9 system mainly includes: physical methods, viral vectors and non-viral vectors. Among them, physical methods are difficult to use in vivo, and viral vectors have potential safety and effectiveness problems. Therefore, many current studies have focused on nonviral vectors.

3.1 Delivery form of the CRISPR / Cas 9 system:

There are three delivery forms of the CRISPR / Cas 9 system.

The first is the complex ribonucleoprotein (ribonucleoprotein, RNP) delivering the Cas 9 protein and the sgRNA. This approach is the most direct, does not require transcription and translation processes, can quickly initiate genome editing, and can significantly reduce off-target effects, toxicity and immune response. The disadvantage is that it is difficult to prepare a large number of highly active Cas 9 protein, and the size of Cas 9 protein is large, making it difficult to deliver RNP effectively.

The second one delivers a mixture of Cas9 mRNA and sgRNA. This approach first yields the mRNA of Cas 9 protein by in vitro transcription and then is transferred intracellularly along with the sgRNA. The mRNA can be translated directly in the cytoplasm and can initiate genome editing relatively quickly.20 The disadvantage is the poor stability of mRNA, and in addition, the transient expression of mRNA in the cell, which limits the duration of gene editing and reduces the effect of gene editing. Therefore, it is difficult to apply these two forms of delivery to the in vivo treatment of the disease.

The third is the CRISPR / Cas 9 system based on the plasmid DNA (plasmid DNA, pDNA) vector, which is also the simplest method. This system encodes the Cas 9 protein and sgRNA with the same vector, thus avoiding multiple transfection of different components. Furthermore, the plasmid-based CRISPR / Cas 9 system has higher stability than that combining Cas9 mRNA and protein. However, the CRISPR / plasmid-based Cas 9 system also has several challenges in tumor treatment, such as the precise and efficient delivery of pDNA to sites of tumorigenesis.

3.2 difficult problem:

1.Off-target effects: CRISPR-Cas 9 may cause genome editing at non-target sites, which can lead to unexpected gene mutations and increase safety risks.
2.Delivery efficiency: Efficient delivery of CRISPR-Cas 9 systems to target cells and tissues is a challenge, especially for some hard-to-reach tissues or cell types.
3.Vector selection: Current delivery of CRISPR-Cas 9 mainly depends on viral and non-viral vectors. Viral vectors, although highly efficient, may elicit an immune response and the risk of integration into the host genome. Non-viral vectors face the problems of delivery efficiency and targeting.
4.Long-term expression and integration risk: If the CRISPR-Cas 9 system is delivered as a plasmid, there is a risk of long-term expression and random integration into the host genome, which could lead to underlying genetic mutations or diseases.
5.Immunogenicity: The Cas 9 protein may trigger a host immune response, which not only affects the therapeutic effect, but may also pose safety concerns.
6.PAM sequence restriction: The Cas 9 enzyme activity depends on the adjacent PAM sequence, which limits the genomic regions that can be targeted by CRISPR-Cas 9.
7.Development of delivery systems: New delivery systems are needed to ensure the efficient, targeted and safe delivery of CRISPR-Cas 9, such as using new delivery methods such as exosomes.

3.3 Targeted CRISPR / Cas 9 delivery

To ensure the effectiveness of the CRISPR / Cas 9 system in genetic manipulation and its widespread application in clinical applications, it is essential to improve its targeting and delivery capabilities. When using viral vector delivery of the CRISPR / Cas 9 system, there is a risk of random insertion into the host cell genome. Non-viral vectors have received much attention for their design flexibility and scalability for large-scale production. Currently, ligand-modified chitosan nanoparticles and extracellular vesicles that can bind to specific ligands significantly enhance the specificity of the CRISPR / Cas 9 system. In addition, stimulus-responsive nanoparticles can be triggered by intracellular signals (e. g., pH, ATP, and glutathione) for more precise gene editing for targeted delivery of the CRISPR / Cas 9 system. Exogenous stimuli-responsive nanoparticles, such as photoresponsive and magnetically responsive nanoparticles, have also shown promising applications.

4. The current solution

4.1 What is outer membrane vesicles(OMVs)?

Gram-negative bacteria-derived outer membrane vesicles (OMVs) , a bilayer spherical nanostructure (100–300 nm) with an internal cavity created into the extracellular milieu, made of the phospholipid bilayer, lipopolysaccharide (LPS), membrane protein, cell wall components, peptidoglycan, ion metabolites, signaling molecules, and nucleic acids .Bacterial pathogen-derived OMVs are enriched with proteins involved in an invasive activity that causes efficient internalization of these vesicles into host cells .[1]

OMVs possess several advantages over traditional drug delivery carriers, including greater drug-loading space and stability, higher biocompatibility, appropriate immunogenicity, and lower cytotoxicity. Despite the intrinsic immune adjuvant properties, the natural anti-tumor effects of them alone are limited. Recently, engineering modifications of OMVs have been developed to endow extracellular vesicles with new properties through genetic engineering or physicochemical methods, aimed at improving the yield, safety, and targeting capability of OMVs during drug delivery.[2]
Based on the advantages of OMVs in delivering drugs, our project plans to utilize probiotic E.coli Nissle 1917 (EcN) as the strain for modification, and construct a more safe and efficient targeted gene editing delivery system.
Escherichia coli Nissle 1917 (EcN) has been used as an active pharmaceutical ingredient in medical products targeting intestinal health for more than 100 years, so using EcN as a strain and modifying it through genetic engineering methods to obtain OMVs as a carrier for Cas9-sgRNA ribonucleoprotein(RNP) has higher safety.
We will display targeting elements on the surface of the EcN membrane, express Cas9-sgRNA RNP within the EcN, and extrude the protoplasts of the EcN through a liposome extruder to obtain engineered OMVs of the desired size carrying Cas9-sgRNA RNP, thereby achieving safer and more efficient targeted gene editing.

4.2 E. coli internal expression of CRISPR/Cas9 RNP

After the work of displaying the targeted elements on the surface of theartificial OMVs membrane, we began to think about how to use this excellent delivery system to achieve the treatment of diseases and other applications.

The premise to achieve this is to design a CRISPR/Cas9 RNP system to express the CRISPR/Cas9 RNP system inside Escherichia coli, and then to verify whether the artificial OMVs cavity packaging soluble protein, that is, whether the CRISPR/Cas9 RNP system selected by us can be effectively packaged into OMVs experimentally. The specific process is as follows (flowchart here).

To express CRISPR/Cas system in EcN, it is necessary to integrate CRISPR/ CAS-related genes into the genome of EcN or transfect plasmids with related genes to construct engineering strains capable of stable inheritance and expression [1]. The application of CRISPR/Cas9 technology to EcN expression system can realize the expression of active RNPs in EcN. By designing specific crRNA and Cas9 proteins, we can achieve customized transformation and optimization of expressed proteins [2].

4.3 Add targeting elements to the surface of E. coli membrane

The CL7 tag is a artificially modified protein. Escherichia coli DNA enzymes CE2, CE7, CE8, and CE9 are a class of endogenous DNA enzymes with similar structures and functions that are highly toxic to Escherichia coli. Escherichia coli contains a DNA enzyme domain with a size of approximately 16 kDa. In Escherichia coli cells, the active domain of Escherichia coli DNA enzyme is inhibited by binding to an inhibitory molecule called an immune protein, which is approximately 10 kDa in size. The binding strength between the domain of Escherichia coli DNA enzyme and immune proteins is close to the strength of covalent bonds. The strong binding ability between Escherichia coli and immune proteins enables strict regulation of its DNA enzyme activity. The dissociation constant Kd between the domain of Escherichia coli E7 DNase (CE7) and its immune protein 7 (Im7) reaches 10-14 to 10-17 M. Im7 contains 87 amino acid residues with a molecular weight of 9.9 kDa. By intercepting the DNA enzyme active domain of CE7 and modifying it, a protein tag called CL7 was obtained. The CL7 tag lost the original DNase activity of CE7 but maintained high affinity with Im7.

In order to add functional components on the surface of ECN, we have modified it and chose to display this technology on the surface. Firstly, the ice nucleation protein anchored on the outer membrane of the cell is expressed using a plasmid and connected to the IM7 protein. Then, functional elements are connected using the CL7 protein. Due to the strong affinity between the Im7 protein and the CL7 tag, when it binds, the target element will be displayed on the outer membrane of the ECN.

4.4 The targeting and shear efficiency of the delivery system were tested in commercial cell lines targeting

Suitable cell lines were selected and cultured to logarithmic growth stage. sgRNA is designed for specific genes of the target cell line to test whether the designed CRISPR/Cas9 gene editing tool has been successfully delivered into the target cell line. The labeled OMVs are co-cultured with the target cell line using the designed CRISPR/Cas9 gene editing artificial OMVs delivery system with specific fluorescent markers. Ensure that the complex can effectively enter the cell interior and observe its binding to the target cell line in cell culture.

Immunofluorescence staining was performed with antibodies against Cas9 protein to observe the distribution of Cas9 in cells. Suitable substrates were selected according to the function of target genes, and comparative experiments were designed to test the targeting of the delivery system.

5. Biosafety

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6. Reference

1. Manghwar, H., Li, B., Ding, X., Hussain, A., Lindsey, K., Zhang, X., & Jin, S. (2020). CRISPR/Cas Systems in Genome Editing: Methodologies and Tools for sgRNA Design, Off-Target Evaluation, and Strategies to Mitigate Off-Target Effects. Advanced science (Weinheim, Baden-Wurttemberg, Germany), 7(6), 1902312.
2. https://doi.org/10.1002/advs.201902312
3. Madigan, V., Zhang, F., & Dahlman, J. E. (2023). Drug delivery systems for CRISPR-based genome editors. Nature Reviews. Drug Discovery, 22(11), 875-894. doi:10.1038/s4
4. Yuan Weixi, Yu Yunmei, Hu Chuncai, Zhao Guoquan. Research progress of CRISPR/Cas9 technology and its improvement measures. Bulletin of Biotechnology, 2017, 33(4): 70-77.
5. Qiao Huanhuan, Zhang Qinghao, Ming Dong. Nanodelivery of CRISPR/Cas9 system and its application in tumor therapy. Progress in Biochemistry and Biophysics 2021,48 (5) : 570~579.
6. Zhao Zixuan, Li Chunhui, Zhou Lili, Zhao Deyao, Weng Yuhua, Xia Xinhua, Huang Yuanyu. Research progress of CRISPR/Cas system delivery technology and its application. Progress in Biochemistry and Biophysics 2020,47 (4) : 286~299.
7. Yimin Dua, Yanfei Liub, Jiaxin Hua, Xingxing Peng, Zhenbao Liua. CRISPR/Cas9 systems: Delivery technologies and biomedical applications. Asian Journal of Pharmaceutical Sciences 18 (2023) 100854.
8. Saba Jalalifar,Hassan Morovati Khamsi, Seyed Reza Hosseini-Fard, Sajad Karampoor,Bahar Bajelan, Gholamreza Irajian,and Rasoul Mirzaei. Emerging role of microbiota derived outer membrane vesicles to preventive, therapeutic and diagnostic proposes. Infect Agent Cancer. 2023; 18: 3.  Published online 2023 Jan 19. doi: 10.1186/s13027-023-00480-4
9. Keshuang Zheng,Yongpu Feng, Lei Li,Fanyang Kong, Jie Gao,and Xiangyu Kong.Engineered bacterial outer membrane vesicles: a versatile bacteria-based weapon against gastrointestinal tumors. Theranostics. 2024; 14(2): 761–787.  Published online 2024 Jan 1. doi: 10.7150/thno.85917.