Human EpCAM gene knockout vector based on CRISPR-Cas9 system and cell strain thereof

Abstract

The invention discloses three human EpCAM gene knockout vectors based on a CRISPR-Cas9 system and cell strains of the human EpCAM gene knockout vectors, and belongs to the technical field of gene engineering and biology. According to the invention, three pairs of sgRNAs (sgRNA-1 and sgRNA-2, sgRNA-3 and sgRNA-4 as well as sgRNA-5 and sgRNA-6) designed based on EpCAM genes are used, a U6 promoter is added in front of each sgRNA, each pair of sgRNAs is linked through T2A, each pair of sgRNAs is respectively connected to a pGMC00010 vector skeleton structure, and three knockout vectors pGMC-KO-EpCAM-1, pGMC-KO-EpCAM-2 and pGMC-KO-EpCAM-3 of the EpCAM genes are obtained. The three recombinant plasmids are respectively transformed into escherichia coli DH5alpha to obtain recombinant microorganisms. The gene knockout vector is used for respectively transfecting HRT-18 cells through a transfection reagent Lipo2000, a puromycin screening monoclonal method is used for obtaining positive cell strains, and finally flow cytometry is used for analyzing and screening to obtain transgenic cells HRT-18-KO-EpCAM-2 and HRT-18-KO-EpCAM-3 cell strains. According to the invention, the carrier with the EpCAM gene knocked out is constructed, and then the colorectal cancer cell strain with the corresponding low-expression EpCAM gene is constructed, which is very important for research on the molecular mechanism of generation and development of EpCAM and colorectal cancer.

Description

Technical Field

[0001] The present invention relates to three human EpCAM gene knockout vectors and cell lines based on the CRISPR-Cas9 system, belonging to the fields of genetic engineering and biotechnology. Background Art

[0002] The most common treatment for early-stage colorectal cancer is surgical resection of the tumor, combined with adjuvant therapies. However, in advanced stages, surgery is often limited to palliative care or to alleviate significant symptoms. With advances in medicine, targeted therapies and immunotherapy are playing an increasingly prominent role in colorectal cancer treatment.

[0003] Epithelial-specific adhesion molecule (EpCAM) is a 40 kDa type I transmembrane glycoprotein that serves as both a colorectal cancer-specific antigen and a tumor stem cell antigen. As a cell adhesion molecule, EpCAM participates in intercellular adhesion and junctions, playing a crucial role in maintaining tissue integrity and function. In colorectal cancer, abnormal expression of EpCAM may weaken intercellular adhesion, promoting tumor cell invasion and metastasis. Furthermore, EpCAM may contribute to the development and progression of colorectal cancer by influencing biological processes such as cell proliferation and apoptosis. In summary, there is a close relationship between EpCAM and colorectal cancer. High EpCAM expression in colorectal cancer is positively correlated with tumor malignancy, and abnormal expression may promote tumor cell invasion and metastasis.

[0004] Although some studies have revealed the relationship between EpCAM and colorectal cancer, many aspects still require further investigation. For example, the specific mechanisms of EpCAM in colorectal cancer require a deeper understanding of the specific mechanisms of EpCAM in the development and progression of colorectal cancer, particularly its differential roles across different subtypes and pathological stages. Research is needed to understand how EpCAM influences biological processes such as proliferation, apoptosis, invasion, and metastasis in colorectal cancer cells, as well as the interactions between these processes. Furthermore, research is needed to explore how EpCAM promotes distant metastasis of colorectal cancer cells by regulating processes such as intercellular adhesion and cell migration. Furthermore, research is needed to investigate how EpCAM influences the interaction between colorectal cancer and microenvironmental components such as immune cells and stromal cells, thereby affecting tumor progression. In summary, the molecular mechanisms of EpCAM and colorectal cancer are a complex and important research area that requires in-depth research and exploration from multiple perspectives. These studies will help us better understand the pathogenesis of colorectal cancer and provide new ideas and methods for its prevention, diagnosis, and treatment. Therefore, in basic research, constructing a vector to knock out the EpCAM gene and then constructing a colorectal cancer cell line with corresponding low expression of the EpCAM gene is very important for studying the molecular mechanism of EpCAM and the occurrence and development of colorectal cancer. Summary of the Invention

[0005] To meet the needs of studying the molecular mechanisms of EpCAM and the occurrence and development of colorectal cancer, the present invention provides three human EpCAM gene knockout vectors and cell lines based on the CRISPR-Cas9 system.

[0006] The first object of the present invention is to provide three human EpCAM gene knockout vectors based on the CRISPR-Cas9 system, the vectors comprising:

[0007] pGMC00010 empty vector backbone structure and sgRNA sequence structure;

[0008] The pGMC00010 empty vector backbone structure includes the gene promoter EF1a Promoter, the target gene Cas9, the marker gene green fluorescent protein RFP for eukaryotic cell screening and puromycin puro. The vector map is as follows: Figure 1 The nucleotide sequence is shown in SEQ ID NO. 4.

[0009] The sgRNA sequence structures are 3 pairs of sgRNAs designed based on the EpCAM gene plus a U6 promoter.

[0010] In one embodiment of the present invention, the nucleotide sequence of the promoter EF1a Promoter of the target gene is shown as SEQ ID NO.5; the nucleotide sequence of the target gene Cas9 is shown as SEQ ID NO.6; the nucleotide sequence of the red fluorescent protein RFP marker gene for eukaryotic cell screening is shown as SEQ ID NO.7; and the nucleotide sequence of puromycin puro is shown as SEQ ID NO.8.

[0011] In one embodiment of the present invention, the nucleotide sequence of U6 promoter + sgRNA-1 + T2A + U6 promoter + sgRNA-2 designed based on the EpCAM gene is shown as SEQ ID NO. 1, the nucleotide sequence of U6 promoter + sgRNA-3 + T2A + U6 promoter + sgRNA-4 designed based on the EpCAM gene is shown as SEQ ID NO. 2, and the nucleotide sequence of U6 promoter + sgRNA-5 + T2A + U6 promoter + sgRNA-6 designed based on the EpCAM gene is shown as SEQ ID NO. 3.

[0012] A second object of the present invention is to provide a method for constructing the above-mentioned gene knockout vector, which comprises the following steps:

[0013] S1. When synthesizing the three gene sequences (U6 promoter + sgRNA-1 + T2A + U6 promoter + sgRNA-2), U6 promoter + sgRNA-3 + T2A + U6 promoter + sgRNA-4, and U6 promoter + sgRNA-5 + T2A + U6 promoter + sgRNA-6), Not1 and EcoR1 restriction sites were added at both ends, respectively. The synthesized sequences were ligated into the empty vector pGMC00010 through these two restriction sites to construct three EpCAM gene knockout vectors (pGMC-KO-EpCAM-1, pGMC-KO-EpCAM-2, and pGMC-KO-EpCAM-3). Figure 2 ).

[0014] S2. Streak the E. coli containing the knockout vector onto an ampicillin-resistant LB plate and culture overnight at 37°C. Isolate several single colonies for sequencing.

[0015] S3. The sequencing results are correct ( Figure 3Monoclonal shake culture and expansion of the above-mentioned monoclonal antibody, extraction of plasmid by endotoxin-free plasmid extraction kit, detection of purity and concentration, and storage at -20℃.

[0016] A third object of the present application is to provide a recombinant microbial cell of the above-mentioned gene knockout vector.

[0017] In an embodiment of the present application, the recombinant microbial cell is a recombinant Escherichia coli.

[0018] In an embodiment of the present application, the recombinant microbial cell is a recombinant Escherichia coli.

[0019] A fourth object of the present application is to provide a transgenic cell of the above-mentioned EpCAM gene knockout vector.

[0020] In an embodiment of the present application, the transgenic cell is a HRT-18-KO-EpCAM-2 cell or a HRT-18-KO-EpCAM-3 cell.

[0021] In an embodiment of the present application, the transgenic cell is a transgenic cell obtained by a method of screening a monoclonal cell after transfecting a HRT-18 cell with a pGMC-KO-EpCAM-2 or a pGMC-KO-EpCAM-3 gene knockout vector.

[0022] A fifth object of the present application is to provide a construction method of the above-mentioned transgenic cell, the method comprising the following steps:

[0023] S1. Transfecting a HRT-18 cell with the above-mentioned pGMC-KO-EpCAM-1, pGMC-KO-EpCAM-2, or pGMC-KO-EpCAM-3 gene knockout vector by transfection reagent Lipo2000.

[0024] S2. Subculturing the transfected cell according to the transfection efficiency at a ratio of 1:10 to 1:45 in area ratio, and subculturing in a 10 mm culture dish.

[0025] S3. According to the results of the puromycin cytotoxicity experiment of the HRT-18 colorectal cancer cell strain, adding the corresponding puromycin content, and screening until the formation of a monoclonal cell strain can be seen.

[0026] S4. Collecting the monoclonal cell subculture with a cloning ring, identifying each monoclonal cell strain by a flow cytometer, and finally obtaining the transgenic cell strain required by the present study.

[0027] The present invention also claims protection for the use of the pGMC-KO-EpCAM-1, pGMC-KO-EpCAM-2, pGMC-KO-EpCAM-3 gene knockout vectors and their recombinant microbial cells, HRT-18-KO-EpCAM-2 cells and HRT-18-KO-EpCAM-3 cells in the fields of biology, medicine and medical treatment. Beneficial effects

[0028] The present invention constructs vectors, recombinant microbial cells and colorectal cancer cell lines that low-express the EpCAM gene, which can knock out or knock down the highly expressed EpCAM gene in eukaryotic cells. This lays a very important foundation for studying the molecular mechanism of the EpCAM gene and the occurrence and development of colorectal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Map of the pGMC00010 empty vector.

[0030] Figure 2 Vector maps of three EpCAM gene knockout vectors: A: pGMC-KO-EpCAM-1; B: pGMC-KO-EpCAM-2; C: pGMC-KO-EpCAM-3.

[0031] Figure 3 Sequencing results of the constructed EpCAM gene knockout vector. A: Sequence alignment of the constructed PGMC-KO-EpCAM-1 recombinant expression vector with sgRNA-1 and sgRNA-2 showed complete consistency; B: Sequence alignment of the constructed PGMC-KO-EpCAM-2 recombinant expression vector with sgRNA-3 and sgRNA-4 showed complete consistency; C: Sequence alignment of the constructed PGMC-KO-EpCAM-3 recombinant expression vector with sgRNA-5 and sgRNA-6 showed complete consistency.

[0032] Figure 4 Flow cytometry detected the expression of EpCAM gene in HRT-18 colorectal cancer cell line, which was 84.60%.

[0033] Figure 5 Screening diagram of EpCAM gene knockout colorectal cancer cell lines. A: Flow cytometry analysis of the HRT-18-KO-EpCAM-1 transgenic colorectal cancer cell line showed EpCAM gene expression of 84.58%; B: Flow cytometry analysis of the HRT-18-KO-EpCAM-2 transgenic colorectal cancer cell line showed EpCAM gene expression of 65.98%; C: Flow cytometry analysis of the HRT-18-KO-EpCAM-3 transgenic colorectal cancer cell line showed EpCAM gene expression of 33.67%. DETAILED DESCRIPTION

[0034] Description of materials in the examples:

[0035] 1. Strain information: Competent Escherichia coli DH5α, a commercial strain.

[0036] 2. Vector information: Plasmid pGMC00010 empty vector is a commercial vector.

[0037] 3.DMEM high-glucose medium, fetal bovine serum (FBS), Opti-MEM medium, PBS buffer, Lipo2000, puromycin, and CD326-PE are all commercial products.

[0038] 4.LB medium formula: 10g / L peptone, 5g / L yeast powder, 10g / L NaCl; add 20g / L agar powder for solid medium.

[0039] Example 1 Construction of EpCAM gene knockout vector

[0040] The human EpCAM gene sequence was searched in NCBI. sgRNA sequences were designed using the CRISPR ERA website. Three pairs were selected based on exon location and preceded by the U6 promoter. Not1 and EcoR1 restriction sites were added to the ends of the synthesized U6 promoter + sgRNA-1 + T2A + U6 promoter + sgRNA-2, U6 promoter + sgRNA-3 + T2A + U6 promoter + sgRNA-4, and U6 promoter + sgRNA-5 + T2A + U6 promoter + sgRNA-6 gene sequences, respectively. These three sequences were ligated into the empty pGMC00010 vector through these two restriction sites to construct the pGMC-KO-EpCAM-1, pGMC-KO-EpCAM-2, and pGMC-KO-EpCAM-3 gene knockout vectors. These three vectors were transformed into competent Escherichia coli DH5α cells and plated on ampicillin-resistant LB solid medium plates for overnight culture at 37°C. Pick several monoclonal clones and send them to the company for sequencing. Figure 3 ) were cultured in LB medium, and the plasmids were extracted using an endotoxin-free plasmid extraction kit. After testing the purity and concentration, the plasmids were stored at -20°C.

[0041] Example 2 Cultivation of colorectal cancer cell line HRT-18

[0042] The culture solution of HRT-18 colorectal cancer cell strain is 90% DMEM high sugar + 10% FBS. The cells to be thawed are taken out from the liquid nitrogen tank and immediately placed in a 37°C water bath kettle, and quickly shaken to melt the frozen cells as much as possible within 1 minute. The frozen tube is quickly moved into the clean bench after alcohol disinfection. The cell solution is moved into a 1.5ml tube with a pipette, centrifuged at 1500rpm / min for 5min, the liquid is discarded, 1ml of culture solution is added to suspend the cells, and the cell counting plate is counted, and the number of cells is 1x10 6 The cells are cultured in a 37°C, 5% CO2 incubator. The cells are subcultured when they are about 90% confluent, and the trypsinization time is about 2min.

[0043] Example 3: Detection of the cytotoxicity of puromycin on colorectal cancer cell strain

[0044] The HRT-18 colorectal cancer cell strain is inoculated in two 24-well plates at a density of 1-2x10 4 The concentration of puromycin is set at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15ng / ul, and 3 replicates are set for each concentration. On the first day, each concentration of puromycin is added, and the medium is changed every 2 days. The lowest concentration at which all cells die within 7-10 days is used as the screening concentration for the transgenic cells of the colorectal cancer cell strain. After detection, the screening concentration of puromycin for HRT-18 is 6ng / ul.

[0045] Example 4: Transfection of colorectal cancer cell strain

[0046] The colorectal cancer cell strain HRT-18 is inoculated in a 24-well plate at a density of 1-2x10 5 When the cells grow to 80% confluence, the serum-free medium is replaced, and the transfection is performed after 2h. Transfection steps: the transfection reagent Lipo2000 and the target plasmid are added to opti-MEM medium (the ratio of plasmid to Lipo2000 is 1:2), and the two liquids are mixed after 5min. After 20min of incubation in the dark, the mixed liquid is added dropwise to the cell culture well. After 6h, the normal culture medium containing serum is replaced, and the transfection efficiency of HRT-18 with the three EpCAM gene knockout vectors is observed under a fluorescence microscope after 18-24h.

[0047] Example 5: Construction of transgenic colorectal cancer cell strain

[0048] Transfected colorectal cancer cells are passaged in 10mm culture dishes at an area ratio of 1:10 to 1:45, depending on the transfection efficiency. Based on the results of the puromycin cytotoxicity test for colorectal cancer cell lines, appropriate puromycin levels are added, and the screening medium is replaced every three days. After 10-14 days of screening, monoclonal cell lines are observed. Cloning rings are used to collect the monoclonal cells and passage them. Once a certain cell number is reached, each monoclonal cell line is identified by flow cytometry to ultimately obtain the desired transgenic cell line.

[0049] Example 6 Flow cytometry analysis and screening of transgenic cell lines

[0050] The non-transgenic HRT-18 colorectal cancer cell line was used as the control group, and the expression of EpCAM gene in the three transgenic cell lines was analyzed by flow cytometry. 6 After 100 cells were grown, the cells were washed twice with PBS to remove the culture medium and serum. After staining with CD326-PE flow cytometry antibody for 30 minutes, the cells were washed twice with PBS to remove the influence of free antibody. The cells were resuspended in PBS and then analyzed by flow cytometry. The expression of EpCAM gene in HRT-18 colorectal cancer cell line was 84.60% ( Figure 4 ), the expression of EpCAM gene in HRT-18-KO-EpCAM-1 transgenic colorectal cancer cell line was 84.58% ( Figure 5 A), the expression of EpCAM gene in HRT-18-KO-EpCAM-2 transgenic colorectal cancer cell line was 65.98% ( Figure 5 B), the expression of EpCAM gene in HRT-18-KO-EpCAM-3 transgenic colorectal cancer cell line was 33.67% ( Figure 5 C). The test results indicated that the HRT-18-KO-EpCAM-1 transgenic colorectal cancer cell line was a false-positive cell line. The cell line was passaged and cryopreserved, ultimately establishing the HRT-18-KO-EpCAM-2 and HRT-18-KO-EpCAM-3 cell lines, which both exhibit low EpCAM gene expression.

[0051] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A eukaryotic gene knockout vector, characterized in that: Three pairs of sgRNAs designed based on the EpCAM gene (sgRNA-1 and sgRNA-2, sgRNA-3 and sgRNA-4, sgRNA-5 and sgRNA-6) were designed. A U6 promoter was added in front of each sgRNA, and each pair was linked with T2A. Finally, they were ligated into the pGMC00010 empty vector backbone structure, respectively, to obtain three EpCAM gene knockout vectors pGMC-KO-EpCAM-1, pGMC-KO-EpCAM-2, and pGMC-KO-EpCAM-3; the nucleotide sequence of U6 promoter + sgRNA-1 + T2A + U6 promoter + sgRNA-2 designed based on the EpCAM gene is shown in SEQ ID NO. 1, the nucleotide sequence of U6 promoter + sgRNA-3 + T2A +U6 promoter + sgRNA-4 designed based on the EpCAM gene is shown in SEQ ID NO. 2, and the nucleotide sequence of U6 promoter + sgRNA-5 + T2A + U6 promoter + The nucleotide sequence of sgRNA-6 is shown in SEQ ID NO. 3, and the nucleotide sequence of the pGMC00010 empty vector is shown in SEQ ID NO.

4.

2. The knockout vector for three EpCAM genes according to claim 1, wherein: The three pairs of sgRNAs designed based on the EpCAM gene were respectively connected between the multiple cloning sites Not1 and EcoR1 of the empty vector pGMC00010, and expressed at a high level under the drive of the U6 promoter.

3. The knockout vector for three EpCAM genes according to claim 1, wherein: The vector contains the Cas9 gene structure and the marker gene RFP gene structure and puro gene structure for transgenic cell screening, which are expressed at a moderate level under the drive of the EF1α promoter.

4. Three recombinant microbial cells, characterized in that: A knockout vector containing the three EpCAM genes according to any one of claims 1 to 3.

5. The recombinant microbial cell according to claim 4, wherein The recombinant microbial cell uses Escherichia coli DH5α as a host.

6. The recombinant microbial cell according to claim 5, wherein The preparation method of the recombinant microbial cells is to connect three pairs of sgRNAs (sgRNA-1 and sgRNA-2, sgRNA-3 and sgRNA-4, sgRNA-5 and sgRNA-6) designed based on the EpCAM gene with a U6 promoter between the multiple cloning sites Not1 and EcoR1 of the pGMC00010 empty vector respectively to obtain three recombinant plasmids pGMC-KO-EpCAM-1, pGMC-KO-EpCAM-2, and pGMC-KO-EpCAM-3, and transform the three recombinant plasmids into Escherichia coli DH5α respectively.

7. A eukaryotic transgenic cell, characterized in that: A knockout vector containing the three EpCAM genes according to any one of claims 1 to 3.

8. The eukaryotic transgenic cell according to claim 7, wherein The eukaryotic transgenic cells are the colorectal cancer cell line HRT-18 as the object of gene editing.

9. The eukaryotic transgenic cell according to claim 8, wherein The pGMC-KO-EpCAM-1, pGMC-KO-EpCAM-2, and pGMC-KO-EpCAM-3 gene knockout vectors were respectively transfected into the colorectal cancer cell line HRT-18 using Lipo2000 transfection reagent, and then monoclonal clones were screened to obtain transgenic cells.

10. Use of a knockout vector containing the EpCAM gene according to any one of claims 1 to 3, or a recombinant microbial cell according to any one of claims 4 to 6, or a eukaryotic transgenic cell according to any one of claims 7 to 9 in the fields of biology, medicine, and healthcare.

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