LentiCRISPRv2 / Cas9-sgRNAs gene editing system based on cancer promoting gene Cebpg as well as preparation method and application of LentiCRISPRv2 / Cas9-sgRNAs gene editing system

Abstract

The invention relates to the field of tumor molecular biology, and particularly discloses a LentiCRISPRv2 / Cas9-sgRNAs gene editing system based on a cancer promoting gene Cebpg as well as a preparation method and application of the LentiCRISPRv2 / Cas9-sgRNAs gene editing system. Aiming at the current situation that CEBPG high expression in gastric cancer is related to tumor progression, a LentiCRISPRv2-Cas9 plasmid is connected with sgRNA of a specific targeting CEBPG gene, and an efficient gene editing system is constructed. The preparation method comprises the following steps: designing an sgRNA sequence of a targeted CEBPG coding region, carrying out single-chain sgRNA annealing, carrying out vector enzyme digestion and connection, preparing a LentiCRISPRv2 / Cas9-sgRNAs vector, and carrying out lentivirus packaging to realize gene knockout. The system can significantly inhibit CEBPG expression, slow down gastric cancer cell proliferation and improve an immunosuppressive microenvironment, and is especially suitable for CEBPG high-expression gastric cancer treatment.

Description

Technical Field

[0001] The present invention relates to the field of tumor molecular biology, and in particular to a LentiCRISPRv2 / Cas9-sgRNAs gene editing system based on the oncogene Cebpg, as well as a preparation method and application thereof. Background Art

[0002] Gastric cancer has a high incidence and mortality rate. Most patients are already in the advanced stage at the time of diagnosis and cannot be cured. Existing chemoradiotherapy and targeted therapy have limited efficacy, single-target drugs are prone to drug resistance, and the 5-year survival rate is less than 30%. There are currently no treatment strategies for new targets such as CEBPG. CEBPG is a bZIP transcription factor. Studies have shown that CEBPG is significantly upregulated in esophageal squamous cell carcinoma (ESCC), activating the PI3K-AKT pathway to promote cancer cell proliferation and migration; in ovarian cancer, it upregulates the ferroptosis inhibitor SLC7A11 to inhibit cell ferroptosis. Preliminary experimental results show that CEBPG expression is elevated in gastric cancer tissues, which may participate in tumor progression by reshaping the immune microenvironment.

[0003] Given the critical role of CEBPG in tumorigenesis and progression, gene editing intervention is crucial. CRISPR / Cas9 technology, with its simple design and high efficiency, has become a crucial tool for precise genome editing. This technology uses sgRNA to guide Cas9 to create a targeted double-strand break in the genome, enabling knockout or regulation of target genes. However, existing technologies still face limitations in editing efficiency and applicability, primarily due to the following: Early lentiviral vectors had low viral titers and required pre-expression of Cas9 in recipient cells, limiting their application in various somatic cell types. Traditional vector construction is cumbersome, making it difficult to rapidly and efficiently prepare editing systems containing specific sgRNA sequences. The improved LentiCRISPRv2 vector significantly increases viral titers, but random insertion of integrating vectors into the host genome carries the potential risk of oncogenicity. Furthermore, sustained expression of the Cas9 nuclease increases the incidence of off-target mutations. The large size of the Cas9 / sgRNA complex makes it difficult for existing viral or nanovectors to achieve both efficient delivery and targeting specificity. Furthermore, exogenous vectors can induce immune responses and be associated with off-target effects.

[0004] In summary, existing gastric cancer treatment methods and gene editing technologies are difficult to meet the needs of efficient targeted intervention for CEBPG. Summary of the Invention

[0005] The purpose of the present invention is to address the shortcomings of the prior art and propose a LentiCRISPRv2 / Cas9-sgRNAs gene editing system based on the oncogene Cebpg, its preparation method and application. To achieve the above purpose, the embodiments of the present invention adopt the following technical solutions:

[0006] In a first aspect, an embodiment of the present invention proposes a method for preparing a LentiCRISPRv2 / Cas9-sgRNAs gene editing system based on the oncogene Cebpg, comprising the following steps: connecting a LentiCRISPRv2-Cas9 plasmid to an sgRNA that specifically targets the Cebpg gene.

[0007] Preferably, the method comprises the following steps:

[0008] Step 1: Design sgRNA targeting the coding region of Cebpg gene according to the target gene sequence, and anneal the single-stranded sgRNA to form a double-stranded sgRNA;

[0009] Step 2: Enzyme digestion of the LentiCRISPRv2 / Cas9 vector;

[0010] In step 3, the vector fragment recovered after enzyme digestion is ligated with the double-stranded sgRNA obtained in step 1 to obtain the LentiCRISPRv2 / Cas9-sgRNAs vector.

[0011] Preferably, the method specifically includes: targeting the target gene Cebpg, using the CRISPR design tool to select the three sgRNA sequences with the highest scores: sgRNA1, sgRNA2 and sgRNA3, and the primer sequences are as follows:

[0012] sgRNA1-F 5*-caccgATATGGCGGTGAAAAAAAGC-3

[0013] sgRNA1-R 5*-aaacGCTTTTTTTCACCGCCATATc-3

[0014] sgRNA2-F 5*-caccgAATACCGCCAGCGCAGAGAG-3

[0015] sgRNA2-R 5*-aaacCTCTCTGCCGCTGGCGGTATTc-3

[0016] sgRNA3-F 5*-caccgAATAAGTGTCATTCATACTC-3

[0017] sgRNA3-R 5*-aaacGAGTATGAATGACACTTATTc-3.

[0018] Preferably, the single-stranded sgRNA is annealed to form a double-stranded sgRNA; the LentiCRISPRv2 / Cas9 plasmid is digested with enzymes; the LentiCRISPRv2 / Cas9 plasmid fragment recovered by enzyme digestion is ligated to the annealed double-stranded sgRNA using T4 DNA ligase, and the resulting ligation product is used to transform DH5α Escherichia coli. After resistance screening, single clones are picked and sequenced to determine whether the sgRNA has been successfully inserted into the LentiCRISPRv2 / Cas9 vector.

[0019] Preferably, a lentiviral packaging step is included: the LentiCRISPRv2 / Cas9-sgRNAs plasmid is co-transfected with viral packaging plasmids psPAX2 and pMD2.G into packaging cells, and PEI is used as a transfection reagent.

[0020] In a second aspect, an embodiment of the present invention proposes a LentiCRISPRv2 / Cas9-sgRNAs gene editing system, comprising a LentiCRISPRv2 / Cas9 lentiviral vector and at least one sgRNA sequence specifically targeting the Cebpg gene.

[0021] In a third aspect, an embodiment of the present invention proposes an application of a LentiCRISPRv2 / Cas9-sgRNAs gene editing system for preparing a tumor treatment preparation.

[0022] Preferably, the tumor is gastric cancer with high Cebpg expression.

[0023] Preferably, the invention is used for preparing a preparation for inhibiting Cebpg expression.

[0024] Preferably, the therapeutic formulation comprises LentiCRISPRv2 / Cas9-sgRNA1, LentiCRISPRv2 / Cas9-sgRNA2;

[0025] or a combination of LentiCRISPRv2 / Cas9-sgRNA1 and LentiCRISPRv2 / Cas9-sgRNA2;

[0026] Preferably, the therapeutic agent is LentiCRISPRv2 / Cas9-sgRNA2.

[0027] Beneficial effects: The embodiment of the present invention provides a simple and efficient method for preparing a gene editing system. By connecting the LentiCRISPRv2-Cas9 plasmid with an sgRNA that specifically targets Cebpg, the problems of cumbersome and low efficiency of the traditional vector construction process are overcome, and a highly specific gene editing tool can be quickly prepared, which is suitable for targeted knockout of the CEBPG gene in gastric cancer, filling the gap in the existing technology for the lack of efficient CEBPG targeted intervention methods. The gene editing system provided by the present invention utilizes the LentiCRISPRv2 / Cas9 lentiviral vector in combination with a specific sgRNA sequence to achieve efficient knockout of the CEBPG gene. Compared with traditional vectors, it significantly improves the viral titer and gene integration efficiency, reduces the risk of off-target mutations, is suitable for long-term stable expression in a variety of somatic cells, and provides an effective molecular tool for the precise treatment of CEBPG-highly expressing tumors such as gastric cancer. The present invention applies the LentiCRISPRv2 / Cas9-sgRNAs gene editing system to prepare tumor therapeutic preparations. Targeting gastric cancer with high CEBPG expression, the invention precisely knocks out the CEBPG gene, reshapes the immune microenvironment, and inhibits tumor growth. This overcomes the drug resistance problem of existing chemoradiotherapy and single-target targeted therapy, provides new treatment hope for gastric cancer patients, and has significant clinical application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Various other advantages and benefits will become apparent to those skilled in the art upon reading the detailed description of the preferred embodiment below. The accompanying drawings are for illustration purposes only and are not to be considered as limiting the present invention. The same reference numerals are used throughout the accompanying drawings to denote the same components. In the accompanying drawings:

[0029] Figure 1 CEBPG is upregulated in gastric cancer tissues and negatively correlated with the prognosis of tumor patients; (AB) Figures show that the expression level of CEBPG in human gastric cancer tissue samples detected by immunohistochemistry is positively correlated with the progression stage of gastric cancer; (C) Figure shows that the expression level and proportion of CEBPG expressing cells are also positively correlated with the progression stage of gastric cancer, *, P<0.05; **, P≤0.01; ***, P≤0.001; ****, P≤0.0001.

[0030] Figure 2 To construct a mouse Cebpg knockout gastric cancer cell line and a C57 mouse orthotopic gastric tumor model; (A) Western Blot analysis of Cebpg knockout efficiency in the mouse gastric cancer cell line, (B) the orthotopic gastric tumor formation process in mice, mouse Cebpg knockout gastric cancer cells were injected into the orthotopic serosa of the stomach of 6-week-old C57 mice. In vivo imaging of the small animals was performed on days 1, 7, 14, and 21, and samples were taken and photographed on day 21.

[0031] Figure 3 Knockout of Cebpg in mouse gastric cancer cells significantly inhibited the in situ tumorigenesis of gastric cancer cells in C57 mice; (AB) Figures show in vivo fluorescence imaging and statistical results of mice; (CE) Figure shows gastric tissue samples taken from mice on day 21, and tumor weight and volume statistics; *, P < 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

[0032] Figure 4 To knock out Cebpg in mouse gastric cancer cells, the formation of an immunosuppressive microenvironment is promoted; (AB) Figures show flow cytometry detection of Cebpg knockout in mouse gastric cancer cells, which leads to increased expression of the CD8+T cell effector marker IFNγ and decreased expression of exhaustion markers (PD-1, Tim3) in the gastric cancer immune microenvironment. DETAILED DESCRIPTION

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the present invention will be briefly introduced below in conjunction with the drawings and the description of the embodiments or the prior art. Obviously, the following description of the structures of the drawings is only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative work. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention.

[0034] Gastric cancer is a common malignant tumor worldwide, with extremely high morbidity and mortality rates in my country and globally. Most gastric cancer patients are diagnosed in the advanced stage or with distant metastases, making radical surgery ineligible. Current clinically used chemoradiotherapy is ineffective, while traditional molecular targeted therapies, due to their single target, are prone to drug resistance, resulting in an extremely poor prognosis for gastric cancer patients, with a 5-year survival rate of less than 30%.

[0035] The development and progression of gastric cancer are closely linked to multiple factors, including genetics, environmental factors, inflammation, and the immune microenvironment. Therefore, in-depth research into its early pathogenesis and the search for new targets for precision immunotherapy are of great significance.

[0036] CEBPG (CCAAT / enhancer binding protein gamma) (HGNC:1837 Entrez gene:1054 Ensembl:ENSG00000153879 MIM:138972 UniProtKB: P53567) is an important transcription factor belonging to the bZIP (basic leucine zipper) family. It was first discovered in the 1990s. Its primary function is to participate in biological processes such as cell proliferation, differentiation, and stress response by regulating gene expression. Aberrant expression of the CEBPG gene is found in various tumors and is closely associated with their malignant biological behavior, but its role varies across tumor types.

[0037] In esophageal squamous cell carcinoma (ESCC), CEBPG expression is significantly upregulated, and this upregulation is associated with an increase in gene copy number. High CEBPG expression is associated with a poor prognosis in ESCC patients, suggesting that it may act as a promoter of tumor progression. Studies have found that CEBPG promotes the proliferation and migration of ESCC cells by activating the PI3K-AKT signaling pathway, thereby accelerating tumor progression. The specific mechanism is that CEBPG directly binds to the distal enhancer and / or promoter regions of genes involved in the PI3K-AKT signaling pathway (such as CCND1, MYC, and CDK2), regulating the expression of these genes and thereby activating the signaling pathway.

[0038] In ovarian cancer (OC), CEBPG also shows high expression and is associated with poor prognosis. CEBPG inhibits ferroptosis by transcriptionally regulating SLC7A11 (solute carrier family 7 member 11), thereby promoting the progression of ovarian cancer. SLC7A11 is a key negative regulator of ferroptosis, which inhibits ferroptosis by transporting extracellular cystine into the cell and converting it into glutathione (GSH). CEBPG binds to the SLC7A11 promoter region, upregulating its expression and inhibiting ferroptosis, thereby promoting the proliferation and migration of ovarian cancer cells.

[0039] In acute myeloid leukemia (AML), abnormal expression of CEBPG is associated with tumor cell differentiation arrest. Studies have shown that dysregulation of CEBPG leads to differentiation arrest in AML cells, thereby maintaining the malignant phenotype of tumor cells. Furthermore, CEBPG is associated with a variety of other diseases, such as chronic obstructive pulmonary disease (COPD) and drug addiction.

[0040] Although the role of CEBPG in various tumors has been extensively studied, its role in gastric cancer remains unclear. Gastric cancer is a common malignancy worldwide, with high morbidity and mortality. Previous studies have found that CEBPG expression levels in gastric cancer tissue are significantly higher than in normal gastric mucosal tissue, suggesting that it may play an important role in the occurrence and progression of gastric cancer. Furthermore, CEBPG may promote tumor progression by reshaping the gastric cancer immune microenvironment, promoting CD8 T cell exhaustion, and inhibiting CD8 T cell cytotoxicity.

[0041] Example 1

[0042] Given the key regulatory role of CEBPG in various malignant tumors, the embodiments of the present invention aim to address the problem of the lack of efficient and specific treatment methods targeting the CEBPG gene in existing gastric cancer treatments, and provide a targeted treatment strategy based on the LentiCRISPRv2 / Cas9 gene editing system to achieve precise knockout of the CEBPG gene in gastric cancer cells and inhibit tumor growth.

[0043] CRISPR / Cas9 (Clustered regularly interspaced palindromic repeats) is a gene-editing technology that directs the Cas9 nuclease to specific DNA sequences, enabling precise genome modification and thereby regulating gene expression. Its mechanism involves the design of a specifically designed single-guide RNA (sgRNA). Upon binding to the Cas9 protein, the sgRNA complements the target DNA, precisely directing Cas9 to the target site. Cas9 then cleaves the DNA at that site, creating a double-strand break (DSB). The cell's DNA repair machinery attempts to repair this break. Non-homologous end joining (NHEJ) can cause mutations, while homology-directed repair (HDR) allows for precise insertion or replacement of gene fragments. CRISPR / Cas9 boasts high target specificity and editing efficiency, making it applicable to gene function research, disease modeling, gene therapy, and biotechnology breeding. In biomedical research, it can be used to reveal gene function, validate drug targets, and develop novel gene therapy strategies.

[0044] Gene editing technology mediated by viral vectors has attracted much attention in recent years. The use of viral vectors can solve problems such as low plasmid transfection efficiency, unstable effects, and the inability to transfect certain types of cells, thereby expanding the application scope of RNA editing technology. Compared with adenoviral vectors and retroviral vectors, lentiviral vectors have unique advantages in the field of gene editing treatment of human diseases, especially in terms of infection of non-dividing cells, genome integration and long-term stable expression, low risk of insertion mutations, and large gene loading capacity, making it a highly promising tool in gene therapy without generating any effective cellular immune response.

[0045] In the examples of the present invention, LentiCRISPRv2 / Cas9 gene editing technology was used to specifically target and knock out Cebpg. The results showed that under the guidance of sgRNA1 and sgRNA2, the LentiCRISPRv2 / Cas9 system can specifically knock out the Cebpg gene in gastric cancer cells, demonstrating the effectiveness of this system in editing Cebpg expression in gastric cancer. In vitro and in vivo experimental results showed that this gene editing system can significantly inhibit the anti-tumor immune effect of gastric cancer cells. These results suggest that the construction of the LentiCRISPRv2 / Cas9-Cebpg-sgRNAs gene editing system and the preparation of tumor treatment preparations can be used for clinical targeted treatment of patients with high Cebpg expression, and are expected to become an important molecular strategy for personalized tumor treatment.

[0046] It was confirmed that CEBPG expression is increased in gastric cancer tissues and is positively correlated with the progression stage of gastric cancer;

[0047] Previous studies have found that CEBPG has been confirmed to be highly expressed in a variety of tumors and plays a role in promoting cancer. To provide strong evidence, samples were collected from 5 non-cancerous patients, 4 intestinal metaplasia (IM), and 4 advanced gastric cancer patients, representing the three typical stages of intestinal gastric cancer progression. Immunohistochemical experiments found that the expression level of CEBPG in gastric cancer patient tissues was significantly higher than that in non-cancerous patient tissues, and was positively correlated with the stage of gastric cancer progression ( Figure 1 AB), and combined with the results of single-cell sequencing, it was found that as gastric cancer progressed, the expression level and cell ratio of CEBPG gradually increased ( Figure 1 C), consistent with the immunohistochemical results. These results suggest that high CEBPG expression may serve as a potential molecular marker for assessing the malignant progression of gastric cancer, and that targeted knockout of CEBPG expression may be a promising molecular strategy for targeted therapy in gastric cancer patients.

[0048] The non-cancerous patient tissues and gastric cancer patient tissue specimens used in the examples of the present invention were obtained from the TCGA database and patients of the PLA Army Specialty Medical Center. The collection of all experimental tissue samples was authorized by the Ethics Committee of the Army Medical University and the consent of the patients was obtained.

[0049] Immunohistochemical staining and gene protein level scoring

[0050] (1) Cut the paraffin slices into 2 μm thick sections and bake them in a 65°C oven for about 2.5 hours.

[0051] (2) Immerse the paraffin sections in xylene (15 min twice), anhydrous ethanol (15 min twice), 95% ethanol (5 min once), 85% ethanol (5 min once), and tap water (2 min once).

[0052] (3) After dewaxing, immerse the sections in distilled water for a few seconds.

[0053] (4) Immerse the sections in an antigen retrieval box filled with sodium citrate or EDTA. Prepare a pressure cooker, add an appropriate amount of water, and place the retrieval box in the boiling pressure cooker. After the water vapor comes up, heat it on medium-high heat for 3 minutes. After the pressure cooker returns to normal pressure, remove the retrieval box and place it in a room temperature environment to cool.

[0054] (5) Wash the repair box with PBS for 5 minutes, repeat 3 times. Use an immunohistochemistry pen to circle the appropriate area around the tissue, add 3% H2O2 to the tissue, shake gently to completely cover the tissue, and incubate at room temperature for 15 minutes.

[0055] (6) Wash the repair box with PBS for 5 minutes, repeat 3 times, wipe the liquid on the slice with paper, add goat serum blocking solution to the tissue, and incubate in a 37℃ incubator for 1 hour.

[0056] (7) Add a few drops of diluted antibody to the tissue and place it in a 4°C refrigerator to let it stand overnight.

[0057] (8) Wash with PBS three times, 5 minutes each time. Depending on the antibody, you can selectively add enhancement solution to the tissue and incubate it in a 37°C incubator for 20 minutes.

[0058] (9) Use paper to remove the enhancement solution, wash with PBS three times, 5 minutes each time, then add secondary antibody to the tissue, shake gently to completely cover the tissue, and incubate in a 37°C incubator for 30 minutes.

[0059] (10) Remove the secondary antibody with paper, wash with PBS for 5 minutes, and repeat 3 times. Add DAB coloring solution to the tissue, observe the color change of the tissue under a microscope, and stop the color development with clean water according to the degree of staining.

[0060] (11) Quickly immerse the slice in the antigen retrieval box containing hematoxylin stain, take it out quickly after 1-2 seconds, and immerse it repeatedly in clean water.

[0061] (12) Immerse the slices in saturated lithium carbonate solution for 15 seconds and stop the reaction with clean water.

[0062] (13) Immerse the sections in 75% alcohol (1 min once), 85% alcohol (1 min once), 95% alcohol (1 min once), and anhydrous ethanol (2 min once) in sequence to dehydrate and make them transparent.

[0063] (14) After the slices are dry, use the tip of a gun to dip a small amount of neutral resin diluted with xylene (neutral resin: xylene = 1:1) and apply it to the tissue. Seal the slices with a coverslip to avoid bubbles. Let them dry and store.

[0064] (15) Two experimenters performed double-blind scoring, and the mean value was used to quantify the protein expression of the target gene in intestinal gastric cancer tissue. Protein expression scoring rule: the product of staining intensity and the percentage of positive cells.

[0065] Table 1. Scoring rules for protein expression in immunohistochemical staining

[0066] Staining intensity score Score Positive cell percentage Score No coloring 0 < 5% 0 Weak positive (yellow) 1 6% ~ 25% 1 Positive (brown) 2 26% ~ 50% 2 Strong positive (dark brown) 3 51% ~ 75% 3 > 75% 4

[0067] Example 2

[0068] Construct and validate the LentiCRISPRv2 / Cas9-Cebpg-sgRNAs gene editing system;

[0069] The LentiCRISPRv2 / Cas9-Cebpg-sgRNAs gene editing system was successfully constructed through annealing-enzyme digestion-ligation-transformation. 293T cells were used to package lentivirus and infect the mouse gastric cancer cell line TKM. Western Blot experiments showed that sgRNA1 and sgRNA2 had a good knockout effect ( Figure 2 A).

[0070] LentiCRISPRv2 / Cas9-Cebpg-sgRNAs vector construction

[0071] (1) The sgRNA primers used were designed on the website http: / / crispr-era.stanford.edu / . The primer synthesis work was commissioned by sending an email to Shanghai Sangon Biotechnology Co., Ltd. for synthesis and delivery. The specific sequences are shown in the technical proposal.

[0072] (2) Enzyme digestion of the LentiCRISPR-V2 vector using the following enzyme digestion system:

[0073] Table 2 sgRNA annealing system

[0074] Reagents Volume (total volume 10ul) LentiCRISPR-V2 5ug BsmBI (NEB : R0580S ) 2ul 10 x NEB Buffer 3.1 10ul dd H2O X ul

[0075] (3) Prepare 1% agarose nucleic acid gel and separate the plasmid fragments by nucleic acid electrophoresis. Set the voltage to 110 V and the time to 28 min.

[0076] (4) Use the Omaga gel recovery kit to excise the gel to recover the digested plasmid, and measure the plasmid concentration and purity using an ultra-micro spectrophotometer.

[0077] (5) Annealing of the three sgRNA oligonucleotide chains:

[0078] Table 3 sgRNA annealing system

[0079] Reagents Volume (total volume 10ul) oligo 1 1 oligo 2 1 10x T4 Ligation buffer (NEB) 1 T4 PNK (NEB) 0.5 Nuclease-Free ddH2O 6.5

[0080] Place the sample into the PCR instrument and set the program according to the following steps.

[0081] Table 4 sgRNA annealing program

[0082] step temperature time 1 37℃ 30min 2 95℃ 5min 3 The temperature drops by 0.1°C every second, and a cycle is formed when the temperature drops by 5°C. Xmin 4 to 12℃ 5min 5 4℃ Maintain for a long time

[0083] (6) Connect the annealed sgRNA product and the digested vector and react at 25°C for 1 hour.

[0084] Table 5 Connection system

[0085] Reagents Usage amount (total volume 10ul) Vector after enzyme digestion 25ng Annealed sgRNA product 0.5ul T4 DNA Ligase 0.5ul 10× T4 Ligation Buffer 1ul Nuclease-Free ddH2O Up to 10ul

[0086] (7) Transformation experiment:

[0087] ① Take competent cells and thaw on ice.

[0088] ② Add 4 μl of the ligated LentiCRISPRv2 / Cas9-Cebpg-sgRNAs plasmid to the competent cells, flick gently to mix, and let stand on ice for 30 minutes.

[0089] ③ Heat shock the competent cells in a 42°C metal bath for 60 seconds.

[0090] ④ Immediately place on ice and let stand for 5 minutes.

[0091] ⑤ Centrifuge at 5000 rpm for 3 minutes at room temperature, discard the supernatant, blow evenly, spread on LB ampicillin resistance agarose plate, invert and culture in a 37℃ incubator overnight.

[0092] (8) Pick three single clones from each plate, add 5 ml of LB broth medium, and culture on a shaker at 37°C for 6-8 hours. Aliquot approximately 20 μl of the bacterial solution and send it for Sanger sequencing to determine whether the sgRNA sequence product is correctly connected to the vector plasmid.

[0093] Lentiviral packaging and infection

[0094] (1) Cultivate 293T cells in a 6 cm culture dish until the density reaches 50-60%, and then use them to package lentivirus.

[0095] (2) pSPAX2 = 2ug; pMD2G = 1ug; target plasmid = 4ug; PEI 14ul, add 250ul serum-free DMEM culture medium in turn, pipette to mix well, and let it stand for 20min.

[0096] (3) Replace the 293T cell culture medium with serum-free and antibiotic-free DMEM.

[0097] (4) After the reaction is completed, add the lentiviral packaging system evenly to the 293T cells and shake gently.

[0098] (5) 12-14 hours after transfection, replace the packaging system with 10% FBS DMEM medium.

[0099] (6) Collect the lentiviral supernatant after 48 h and 72 h, filter it with a 0.45 μm filter, and store it in a 4°C refrigerator.

[0100] (7) Infection of mouse gastric cancer cell line: The mouse gastric cancer cell line was evenly spread into a six-well plate. After overnight culture, the plate was washed twice with PBS. 1 ml of culture medium or virus solution and 1 ul of polybrene were added. After thorough mixing, the plate was placed in a 37°C constant temperature incubator for culture.

[0101] (8) Change the medium 24 hours after viral infection.

[0102] (9) 48 hours after changing the medium, add 4 μl of antibiotics (puromycin or hygromycin) to each well for screening. Stop screening when the control cells are completely killed.

[0103] (10) Cells were passaged and a portion of the cell extracts were collected for Western Blot verification.

[0104] Western blot assay

[0105] (1) RIPA lysis protein extraction

[0106] ① Add an appropriate amount of RIPA cell lysis buffer (containing protease and phosphatase inhibitors) based on the volume of the cell pellet. Lyse the cells on ice for 30 minutes, vortexing for 15 seconds every 10 minutes. After sufficient lysis, no obvious precipitation should be observed in the cell suspension.

[0107] ② Centrifuge at 12,000 rpm for 10 min at 4°C and transfer the supernatant to a new centrifuge tube.

[0108] (2) Detection of protein concentration by BCA method

[0109] ① After protein extraction with RIPA cell lysis buffer, prepare BSA protein concentration standards with concentrations of 2000ug / ml, 1500ug / ml, 1000ug / ml, 750ug / ml, 500ug / ml, and 0 (blank).

[0110] ② Preparation of BCA working solution: Mix 50 parts of solution A with 1 part of solution B.

[0111] ③ Add 200ul of BCA working solution to each well of a 96-well plate, followed by 25ul of protein standards and test proteins of various concentrations, and set up empty control wells.

[0112] ④ After mixing, incubate in a 37°C incubator for 30 minutes.

[0113] ⑤ Take out the 96-well plate and measure the absorbance at 562 nm using a microplate reader.

[0114] ⑥ Calculate the protein sample concentration based on the standard curve.

[0115] ⑦ Add 5× SDS loading buffer to the protein sample, vortex and mix thoroughly, and denature at 100℃ for 10 min.

[0116] (3) Prepare 12% SDS-PAGE separation gel.

[0117] (4) Western blotting

[0118] ① Spot the sample and add an appropriate amount of protein sample to each well according to the sample concentration.

[0119] ② 80V, 30 minutes; 120V, about 1 hour and 20 minutes.

[0120] ③ Transfer the membrane, constant current 250mA, electroporation for 2h.

[0121] ④ Block the plate by incubating in 5% skim milk powder in PBST solution at room temperature on a shaker for 1-2 hours.

[0122] ⑤ Wash the NC membrane with PBST, add primary antibody, and incubate overnight at 4°C in a shaker.

[0123] ⑥ Wash three times on a shaker at room temperature, 10 min each time.

[0124] ⑦ Incubate with secondary antibody at room temperature on a shaker for 1 hour.

[0125] ⑧ Wash 3 times with PBST on a shaker at room temperature, 10 min each time

[0126] ⑨Image formation by developer.

[0127] Implementation Case 3

[0128] In vivo experiments verified that the LentiCRISPRv2 / Cas9-Cebpg-sgRNAs gene editing system can effectively knock out Cebpg expression and inhibit the malignant progression of gastric cancer

[0129] Mouse gastric orthotopic transplantation tumor experiment

[0130] (1) Eighteen male C57 mice, 6 weeks old and weighing approximately 20 g, were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. They were maintained under specific pathogen-free (SPF) conditions in the animal room of the PLA Army Specialty Medical Center.

[0131] (2) According to the number of mice in each group and the number of cells required to be injected into each mouse, the mouse gastric cancer cell line TKM was used to prepare control group cells (LentiCRISPRv2 / Cas9-sgNC) and experimental group cells. The experimental group was divided into two groups: a group stably expressing LentiCRISPRv2 / Cas9-Cebpg-sgRNA1 and a group stably expressing LentiCRISPRv2 / Cas9-Cebpg-sgRNA2.

[0132] (3) Each group consisted of 6 C57 mice, each containing 1*10 6 25ul of a cell suspension (PBS: Matrigel = 3:2) was prepared and inoculated into the serosa of the mouse stomach using an insulin syringe.

[0133] (4) Live fluorescence imaging of mice was performed on Day 1, Day 7, Day 14, and Day 21 respectively ( Figure 2 B. Figure 3 A).

[0134] GraphPad Prism software was used to draw growth curves of imaging data ( Figure 3 B).

[0135] (5) In accordance with animal ethics requirements, after Day 21 imaging is completed, the mice are killed by cervical dislocation and weighed. The stomach of the mouse is removed, dissected along the greater curvature of the stomach, the stomach contents are cleaned, and photographed and weighed. Figure 3 C).

[0136] (6) GraphPad Prism was used to perform statistical analysis on the data of gastric tumors: mouse weight and tumor volume. Figure 3 As shown: Compared with the control group, the LentiCRISPRv2 / Cas9-Cebpg-sgRNA1 group and the LentiCRISPRv2 / Cas9-Cebpg-sgRNA2 group can significantly inhibit the growth rate of tumors in situ and reduce tumor weight ( Figure 3 DE), and LentiCRISPRv2 / Cas9-Cebpg-sgRNA2 has a better inhibitory effect on tumor in situ growth.

[0137] Flow cytometry analysis to detect changes in the immune microenvironment of gastric cancer

[0138] (1) Place the tumor mass for flow cytometry detection in a 5 ml EP tube and quickly chop it on ice.

[0139] (2) Add 4-5 ml of tissue digestion solution to resuspend, transfer to a 15 ml centrifuge tube, and digest on a shaking platform at 37°C for 30 minutes.

[0140] (3) After digestion, add an equal volume of complete culture medium to terminate digestion, filter the tissue through a 40 mm filter, and centrifuge the filtrate at 500 g at 4°C for 5 min.

[0141] (4) Discard the supernatant, resuspend the cells in 1 ml of PBS and count them.

[0142] (5) Separate 1*107 cells, centrifuge at 500g for 5 minutes, discard the supernatant, resuspend in 100ul PBS, add flow cytometry antibody staining, and divide into full-stained tubes, single-stained tubes, and blank control. Incubate at 4℃ in the dark for 30 minutes.

[0143] (6) Add 1 ml of complete culture medium and centrifuge at 500 g for 5 min at 4°C.

[0144] (7) Discard the supernatant, resuspend, filter through a 40mm filter, transfer to a flow cytometry tube, and wait for the machine to check. The flow cytometry analysis results showed that after knocking out Cebpg, the expression of effector CD8+T cells in the tumor microenvironment was significantly increased, and the expression of exhausted CD8+T cells was inhibited ( Figure 4 AB).

[0145] In summary, the LentiCRISPRv2 / Cas9-Cebpg-sgRNAs gene editing system can significantly inhibit the expression of the Cebpg gene, reprogram the immunosuppressive microenvironment of gastric cancer, and inhibit the malignant progression of gastric cancer. Moreover, this gene editing system has a broad spectrum and can play an anti-cancer role in various tumors such as gastric cancer and breast cancer, providing a basis for precise tumor treatment.

[0146] In short, the above are only preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of the present invention.

Claims

1. A method for preparing a LentiCRISPRv2 / Cas9-sgRNAs gene editing system based on the oncogene Cebpg, characterized in that: The method comprises the following steps: connecting the LentiCRISPRv2-Cas9 plasmid to an sgRNA that specifically targets the Cebpg gene.

2. The preparation method according to claim 1, characterized in that The steps include: Step 1: Design sgRNA targeting the coding region of Cebpg gene according to the target gene sequence, and anneal the single-stranded sgRNA to form a double-stranded sgRNA; Step 2: Enzyme digestion of the LentiCRISPRv2 / Cas9 vector; In step 3, the vector fragment recovered after enzyme digestion is ligated with the double-stranded sgRNA obtained in step 1 to obtain the LentiCRISPRv2 / Cas9-sgRNAs vector.

3. The preparation method according to claim 2, characterized in that Specifically include: For the target gene Cebpg, the three sgRNA sequences with the highest scores were selected using the CRISPR design tool: sgRNA1, sgRNA2, and sgRNA3. The primer sequences are as follows: sgRNA1-F 5*-caccgATATGGCGGTGAAAAAAAGC-3 sgRNA1-R 5*-aaacGCTTTTTTTCACCGCCATATc-3 sgRNA2-F 5*-caccgAATACCGCCAGCGCAGAGAG-3 sgRNA2-R 5*-aaacCTCTCTGCCGCTGGCGGTATTc-3 sgRNA3-F 5*-caccgAATAAGTGTCATTCATACTC-3 sgRNA3-R 5*-aaacGAGTATGAATGACACTTATTc-3.

4. The preparation method according to claim 1, characterized in that The single-stranded sgRNA was annealed to form a double-stranded sgRNA; the LentiCRISPRv2 / Cas9 plasmid was digested with enzymes; the LentiCRISPRv2 / Cas9 plasmid fragment recovered from the digestion was ligated with the annealed double-stranded sgRNA using T4 DNA ligase. The resulting ligation product was used to transform DH5α Escherichia coli. After resistance screening, single clones were picked and sequenced to determine whether the sgRNA was successfully inserted into the LentiCRISPRv2 / Cas9 vector.

5. The preparation method according to claims 1-4, characterized in that The lentiviral packaging step includes: the LentiCRISPRv2 / Cas9-sgRNAs plasmid is co-transfected with the viral packaging plasmids psPAX2 and pMD2.G into packaging cells, and PEI is used as the transfection reagent.

6. A LentiCRISPRv2 / Cas9-sgRNAs gene editing system, characterized in that Contains the LentiCRISPRv2 / Cas9 lentiviral vector and at least one sgRNA sequence specifically targeting the Cebpg gene.

7. The use of the LentiCRISPRv2 / Cas9-sgRNAs gene editing system according to claim 6, characterized in that Used to prepare tumor treatment preparations.

8. The use according to claim 7, characterized in that The tumor is gastric cancer with high Cebpg expression.

9. The use according to claim 7, characterized in that Used for preparing preparations for inhibiting Cebpg expression.

10. The use according to claim 7, characterized in that The therapeutic formulation includes LentiCRISPRv2 / Cas9-sgRNA1, LentiCRISPRv2 / Cas9-sgRNA2; Or a combination of LentiCRISPRv2 / Cas9-sgRNA1 and LentiCRISPRv2 / Cas9-sgRNA2.

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