Sgrna, mdck cells and uses thereof

Abstract

This invention relates to the field of bioengineering, and more particularly to sgRNA, MDCK cells, and their applications. This invention provides sgRNA having the nucleotide sequences shown in SEQ ID NO:1 to SEQ ID NO:4. This invention employs a CRISPR-Cas9 gene editing method, which, compared to existing RNAi methods, offers higher specificity and lower off-target risk. Compared to MDCK cells, PCR detection showed a reduction of over 90% in RNA levels in IRF7- / - cells, Western blotting showed a significant decrease in IRF7 protein expression, and viral hemagglutination assays showed higher titers for influenza virus compared to the original cells. Tumorigenicity assays showed that the gene knockout cells retained low tumorigenicity. Compared to existing technologies, the cells of this invention exhibit both low tumorigenicity and significantly increased viral yield, making them suitable for influenza vaccine production.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, and more particularly to sgRNA, MDCK cells, and their applications. Background Technology

[0002] Large-scale, efficient production of influenza vaccines is a crucial element of global public health systems in responding to seasonal influenza and potential pandemics. Traditional chicken embryo vaccine production technology is limited by factors such as chicken embryo supply constraints, long production cycles, and poor adaptability to certain strains, making it difficult to meet urgent needs. Therefore, vaccine production technology based on mammalian cell culture, especially using MDCK cells as a production matrix, has become an important direction for replacing and upgrading influenza vaccine production processes internationally.

[0003] MDCK cell lines are widely considered the "gold standard" for influenza virus culture due to their high sensitivity and broad susceptibility to influenza viruses. Their advantages include short production cycles, controllable processes, and no limitations imposed by chicken embryos, making them more suitable for meeting the urgent production needs during outbreaks. However, this cell line still faces two major challenges in actual vaccine production: firstly, its inherent strong tumorigenicity poses potential biosafety risks; secondly, the sensitivity of some cell lines to influenza viruses still needs improvement, resulting in lower virus yields (titers) and impacting production costs and economic viability.

[0004] To overcome these challenges, the industry's research focus is on the targeted modification of MDCK cells using genetic engineering techniques. Current technologies are primarily advancing along two lines: first, by knocking out specific genes to reduce cell tumorigenicity. For example, studies have successfully obtained MDCK cell lines with significantly reduced tumorigenicity by knocking out genes such as LC3, SQSTM1, and Bcl-xL; second, by intervening in related genes to improve cell sensitivity to viruses and viral yield. For instance, knocking out the sialyltransferase gene ST3GAL1 can increase sensitivity to human influenza virus, knocking out the FLNB gene can reduce cell adhesion and thus increase viral yield, while knocking out the SLC35B2 gene has been shown to promote adenovirus proliferation.

[0005] Of particular note is the crucial role of interferon regulatory factor 7 (IRF7) as a "master switch" in cellular antiviral innate immune responses. Studies have shown that knocking down IRF7 gene expression in MDCK cells using RNA interference effectively inhibits the interferon signaling pathway, significantly weakening the cells' inherent antiviral state and resulting in a substantial increase in influenza virus replication efficiency (reportedly up to approximately nine-fold). This provides a clear theoretical and practical basis for constructing highly efficient virus-producing cell lines by regulating the host cell's innate immune pathways.

[0006] In recent years, CRISPR-Cas9 gene editing technology has become a key tool for genetic modification due to its high efficiency, precision, and ease of operation, providing strong technical support for the targeted genetic improvement of MDCK cells.

[0007] In summary, although low-tumorigenicity MDCK cell lines can be obtained through techniques such as monoclonal screening, these cells often suffer from insufficient viral sensitivity and low yield. Therefore, how to utilize advanced gene editing technology to further remove or weaken the antiviral immune limitations of MDCK cells (such as targeting the IRF7 pathway) while ensuring low tumorigenicity, thereby constructing a novel cell matrix that combines "high safety" and "high production efficiency," has become a core technical problem that urgently needs to be solved to improve influenza vaccine production capacity and reduce production costs, and has significant industrial application value. Summary of the Invention

[0008] In view of this, the present invention provides sgRNA, MDCK cells, and their applications. The present invention employs a CRISPR-Cas9 gene editing method, which, compared with existing RNAi methods, offers higher specificity and lower off-target risk. Compared to MDCK cells, PCR detection shows that the RNA level in IRF7- / - cells is reduced by more than 50%, Western blotting shows a significant decrease in IRF7 protein expression, and viral hemagglutination assays show a higher titer for influenza virus than the original cells. Tumorigenicity assays show that the gene knockout cells retain low tumorigenicity. Compared with existing technologies, the cells of the present invention possess both low tumorigenicity and significantly increased viral yield, making them suitable for influenza vaccine production.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0010] This invention provides sgRNA, which has the following characteristics:

[0011] (1) Nucleotide sequences as shown in SEQ ID NO:1 to SEQ ID NO:4; or

[0012] (2) A nucleotide sequence obtained by modifying, substituting, deleting, or adding one or more bases to the nucleotide sequence described in (1); or

[0013] (3) A sequence having at least 80% homology to the nucleotide sequence described in (1) or (2); or

[0014] (4) The complementary sequence of the nucleotide sequence described in (1), (2) or (3).

[0015] This invention also provides the application of the above-mentioned sgRNA in knocking out the IRF7 gene in MDCK cells.

[0016] The present invention also provides a method for knocking out the IRF7 gene in MDCK cells, comprising the step of introducing the above-mentioned sgRNA into MDCK cells.

[0017] In some embodiments of the present invention, the above method includes the step of introducing the above-mentioned sgRNA and Cas protein into MDCK cells.

[0018] In some embodiments of the present invention, the molar ratio of sgRNA to Cas protein in the above method is (5~10):1.

[0019] The present invention also provides MDCK cells with the IRF gene knocked out by the above method.

[0020] In some embodiments of the present invention, the above-mentioned MDCK cells have the accession number CCTCC NO:C202610(M16).

[0021] The present invention also provides a primer pair for detecting the above-mentioned MDCK cells, characterized in that it has:

[0022] (5) Nucleotide sequences as shown in SEQ ID NO:5 and SEQ ID NO:6; or

[0023] (6) A nucleotide sequence obtained by modifying, substituting, deleting, or adding one or more bases to the nucleotide sequence described in (5); or

[0024] (7) A sequence having at least 80% homology to the nucleotide sequence described in (5) or (6); or

[0025] (8) The complementary sequence of the nucleotide sequence described in (5), (6) or (7).

[0026] The present invention also provides the use of the above-mentioned MDCK cells in the preparation of products for the treatment and / or prevention of influenza viruses.

[0027] The present invention also provides a product comprising: the above-described MDCK cells.

[0028] In some embodiments of the present invention, the above-mentioned products include vaccines and / or drugs.

[0029] This invention utilizes CRISPR-Cas9 gene editing technology to achieve gene knockout in MDCK cells. The process includes: 1. Designing sgRNA primers. 2. Using the CRISPR-Cas9 gene editing system to knock out the IRF7 gene in MDCK cells. 3. Cell cloning. 4. NGS sequencing to confirm the knockout cell clones and selecting positive cells. 5. NGS technology to verify the gene knockout efficiency of positive cells. 6. Western blotting to detect the IRF7 protein expression level in positive cells. 7. Cell acclimation to achieve serum-free culture. 8. Hemagglutination assays to screen cell sensitivity to various influenza viruses. 9. Tumorigenicity assays to verify cell tumorigenicity. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0031] Figure 1 The qPCR amplification curve of the cell nucleic acid extract is shown;

[0032] Figure 2 Western blot images of total cellular protein extracts are shown; the left side shows the WB results for GAPDH reference protein, and the right side shows the IRF7 protein detection results. Lanes 1-8 are Marker, MDCK-WT, MDCK-OE-5, MDCK-OE-20, MDCK-OE-25, MDCK-OE-27, Yangshen 1 (Jukart), and Yangshen 2 (THP-1), respectively.

[0033] Figure 3 Tumorigenicity assays of cells (from left to right: MDCK (NBL-2), MDCK-25, MDCK-27);

[0034] Figure 4 The differential gene Venn diagram shows the cells (where: ctrl represents MDCK tumorigenic cells, M06 represents MDCK non-tumorigenic cells, and M16 represents gene knockout MDCK cells).

[0035] Figure 5 A statistical graph showing the number of differentially expressed genes in cells;

[0036] Figure 6 Principal component analysis of cells is shown (where: ctrl represents MDCK tumorigenic cells, M06 represents MDCK non-tumorigenic cells, and M16 represents gene knockout MDCK cells).

[0037] Biological Preservation Instructions

[0038] Canine kidney cells MDCK.3.1.6-KO (Canis lupus familiaries), deposited on January 14, 2026, with accession number CCTCC No:C202610, deposited at China Center for Type Culture Collection, Wuhan University, Wuhan, China. Detailed Implementation

[0039] This invention discloses sgRNA, MDCK cells, and their applications.

[0040] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0041] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0042] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0043] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0044] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0045] In Examples 1 to 7 of this invention, all raw materials and reagents used can be purchased from the market.

[0046] The present invention will be further illustrated below with reference to the embodiments:

[0047] Example 1

[0048] Design, synthesis, and sequencing of sgRNA sequences targeting the ORF sequence of the IRF7 gene.

[0049] Table 1

[0050]

[0051] Example 2: Liposome Transfection

[0052] (1) After multiple monoclonal screenings and expansion culture of the MDCK(NBL-2)CCL-34 cell line (purchased from ATCC), the cells were cultured in a medium containing graded concentrations of bovine serum. Non-tumorigenic MDCK cell lines were obtained after tumorigenicity verification. This cell line eliminated the risk of tumorigenicity and significantly improved the biosafety of the cellular matrix influenza vaccine. The strain has the preservation number CCTCC NO:C202504 and a Chinese patent application has been filed, application number: 202610137381.0.

[0053] (2) Cell preparation: Remove the cryovials of low-tumorigenic MDCK cells (internal code M06) from the liquid nitrogen tank or -80℃ freezer, and quickly place the cryovials in a 37℃ water bath, shaking them back and forth to thaw them rapidly. Place the cryovials in a biosafety cabinet, and transfer the liquid in the cryovials to a centrifuge tube containing approximately 9 mL of complete culture medium. Centrifuge at 300g for 5 min, and discard the supernatant. Resuspend the cells in an appropriate amount of complete culture medium and transfer them to a new culture flask. Place the cells in a 5% CO2, 37℃ constant temperature incubator for static culture. When the cell density reaches 80% or higher, passage can be performed at a passage ratio of 1:3 to 1:4, passaged every 2 to 3 days. Aspirate and discard the culture medium in the culture flask, add an appropriate amount of PBS, gently shake to rinse the cells, and then aspirate the PBS. Add approximately 1.0 mL of 0.25% trypsin, gently agitate to infiltrate the cells, and then place in an incubator for digestion for 2-3 minutes. Observe under a microscope until the cells become rounded. Gently tap the flask wall to observe that 80% of the cells have detached from the wall. At this point, add approximately 5 mL of complete culture medium to neutralize the trypsin. Gently pipette to dislodge the cells from the surface of the culture flask, dispersing them into a single-cell suspension. (This is the volume used for a T75 culture flask; adjust the amount according to your actual situation.) Transfer the cell suspension after digestion to a clean 15 mL centrifuge tube, centrifuge at 300 g for 5 minutes, and discard the supernatant. Resuspend the cells in an appropriate amount of complete culture medium and transfer them to a new culture flask according to the passage ratio. Place the cells in a 5% CO2, 37°C incubator for static culture.

[0054] (3) RNP preparation: Prepare the RNP complex at a ratio of 5:1 to 10:1 (sgRNA:Cas9) and let it stand for 10 minutes.

[0055] (4) Electroporation procedure: Use an electroporation instrument to perform RNP nuclear transfection. Refer to the instructions for use of the Lonza and Thermo FisherNeon transfection systems to perform the RNP nuclear transfection experiment.

[0056] Result detection:

[0057] After electroporation, the cells were cultured in culture medium for 24 hours. Monoclonal cells were obtained by limiting dilution. Single clones were selected for qPCR detection. RNA was extracted from MDCK cells and reverse transcribed. Then, qPCR primers were used to simultaneously detect the original expression level of the IRF7 gene in MDCK cells, using GAPDH as a reference gene. The primer design is as follows:

[0058] Table 2

[0059]

[0060] Depend on Figure 1 The qPCR results showed that the IRF7 gene was reduced in monoclonal cells of MDCK-OE-5, MDCK-OE-20, MDCK-OE-25, and MDCK-OE-27, which can be used for further investigation.

[0061] Example 3: Western blot detection of IRF7 protein expression level in monoclonal cells

[0062] Single clones 5, 20, 25, and 27 were selected for Western blotting (WB). Cell samples (including MDCK-WT, MDCK-OE-5, MDCK-OE-20, MDCK-OE-25, MDCK-OE-27, Jukart, and THP-1) were lysed to extract proteins, and then Western blotting was performed to detect the relative expression levels of the target genes at the protein level in the target cells. The results are as follows: Figure 2 As shown, Figure 2 The left side shows the Western blot results for the GAPDH reference protein. Figure 2 The right side shows the IRF7 protein detection results.

[0063] Table 3

[0064]

[0065] Depend on Figure 2 The WB results showed that the GAPDH reference protein was expressed normally in all samples, the IRF7 protein was expressed normally in wild-type cells, its expression was reduced in MDCK-OE-5 and MDCK-OE-20 cells, and it was basically not expressed in MDCK-OE-25 and MDCK-OE-27 cells.

[0066] Example 4: Domestication of monoclonal cells, serum-free culture

[0067] Discard the serum-containing culture medium, wash twice with PBS, add trypsin for 6-10 min, neutralize the trypsin with serum-free medium containing 10% bovine serum, mix well by pipetting, centrifuge at 900 rpm / 4 min, discard the supernatant, resuspend in serum-free medium containing 10% bovine serum, and passage. Each serum-containing medium should be passaged at least twice. Only after the cells are in good growth condition should serum-free medium be passaged again. Passage should be performed in the following manner: 10% bovine serum + serum-free medium → 7.5% bovine serum + serum-free medium → 5% bovine serum + serum-free medium → 2.5% bovine serum + serum-free medium → 0.5% bovine serum + serum-free medium → serum-free medium.

[0068] Example 5: Detection of tumorigenicity of cells

[0069] Soft agar experiment: by Figure 3 It can be seen that the positive control cells MDCK(NBL-2) showed obvious cell clumps on soft agar, while cells No. 25 and No. 27 did not have clumps, indicating that cells No. 25 and No. 27 did not have tumorigenicity on soft agar.

[0070] Nude mice aged 4-7 weeks were selected, with 10 mice in each group. Each mouse was subcutaneously injected with 0.2 mL of the cells to be tested (i.e., each mouse was inoculated with 10...). 7 (10 live cells); the positive control group was injected with 0.2 mL of tumor-positive cells per animal, containing 10 6 Live cells. Regularly observe and palpate all animals at the injection site for nodule formation, for at least 16 weeks. Select cell lines where nodules have regressed and perform further pathological examination to confirm the absence of tumor growth.

[0071] Cell accession number: CCTCC No: C202610, deposit date: January 14, 2026. Deposited at the China Center for Type Culture Collection (Wuhan University Collection Center).

[0072] Example 6: Detection of the susceptibility of monoclonal cells to influenza virus, titer detection.

[0073] Viral adaptability verification: The selected cells (MDCK-OE-25, MDCK-OE-27) were subjected to viral adaptability verification. The virus was diluted 100-100,000 times (or converted to MOI), and trypsin was inoculated at 5μg / mL-15μg / mL into T25 and T75 square flasks and microspheres.

[0074] Virus susceptibility tests were performed on M16 cells using T25 flasks, with the following strains used for validation and comparison with the original tumorigenic cells (Control).

[0075] Strain H1N1: 23 / 250: Influenza Virus Infectious IVR-238 (A / Victoria / 4897 / 2022);

[0076] 16 / 270: Influenza virus infectious IVR-180;

[0077] Strain H3N2: 17 / 196: Influenza Virus Infectious A / Singapore / INFIMH-16-0019 / 2016;

[0078] 21 / 204: Influenza Virus Infectious A / Thailand / 8 / 2022;

[0079] Strain BV: 22 / 204: Influenza Virus Infectious B / Austria / 1359417 / 2021 (B-Victoria lineage)BVR-26;

[0080] 23 / 228:Influenza Virus Infectious B / Austria / 1359417 / 2021 (B / Victoria)

[0081] The results of hemagglutination titer verification in T25 bottles showed that the hemagglutination titer of MDCK-OE-25 and MDCK-OE-27 cells after gene knockout was increased, which significantly improved viral yield.

[0082] Table 4

[0083]

[0084] Example 7 Transcriptome analysis of target cells

[0085] The resulting highly viral-adapted cells, MDCK-OE-27 (numbered M16), along with the starting cells (non-tumorigenic cells, numbered M06) and control cells (tumorigenic cells, Control), were sent to a third-party company for transcriptome analysis. The results are as follows:

[0086] (1) Venn diagram: such as Figure 4 As shown, the three genomes have some overlap, but also exhibit certain genetic differences. PCA analysis ( Figure 6The results further showed that M16, M6 and tumorigenic control cells were completely separated on PC1 and PC2, indicating that the two groups had significant differences in genetic variation.

[0087] (2) Gene upregulation and downregulation

[0088] like Figure 5 As shown, the expression levels of the M06 / M16 genes showed 3347 upregulations and 3225 downregulations. Differentially expressed genes were sorted by their expression difference ratio (log2(M16 / M06)), with the highest and lowest 100 genes selected for GO and KEGG analyses. GO functional enrichment analysis revealed that differentially expressed genes were significantly enriched in multiple items related to viral infection. The most strongly / significantly associated biological pathways were, in descending order: G0:0035458 (cellular response to interferon-β), G0:0052372 (symbiotic regulation of host entry), G0:0046596 (regulation of viral entry into host cells), G0:0060429 (epithelial tissue development), and G0:0009888 (tissue development). KEGG functional enrichment analysis also showed that differentially expressed genes were significantly enriched in multiple items related to viral infection. Among them, the most strongly associated / significant biological pathways were cfa04061 (interaction between viral proteins and cytokines and cytokine receptors), cfa05164 (influenza A), and cfa04721 (synaptic vesicle circulation). This indicates that knocking out the IRF7 gene alters the cells' resistance to viral infection, explaining the possible mechanism of increased viral titers in cell culture.

[0089] Table 5

[0090]

[0091] Table 6

[0092]

[0093] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. sgRNA, characterized by, It has the following characteristics: (1) Nucleotide sequences as shown in SEQ ID NO:1 to SEQ ID NO:4; or (2) A nucleotide sequence obtained by modifying, substituting, deleting, or adding one or more bases to the nucleotide sequence described in (1); or (3) A sequence having at least 80% homology to the nucleotide sequence described in (1) or (2); or (4) The complementary sequence of the nucleotide sequence described in (1), (2) or (3).

2. The application of the sgRNA as described in claim 1 in knocking out the IRF7 gene in MDCK cells.

3. A method for knocking out the IRF7 gene in MDCK cells, characterized in that, include: The steps for introducing sgRNA into MDCK cells as described in claim 1.

4. The method as described in claim 3, characterized in that, include: The steps for introducing sgRNA and Cas protein into MDCK cells as described in claim 1.

5. The method as described in claim 4, characterized in that, The molar ratio of the sgRNA to the Cas protein is (5~10):

1.

6. MDCK cells with the IRF gene knocked out obtained by the method of any one of claims 3 to 5.

7. The MDCK cells as described in claim 6, characterized in that, Its accession number is CCTCC NO:C202610.

8. A primer pair for detecting MDCK cells as described in claim 6 or 7, characterized in that, It has the following characteristics: (5) Nucleotide sequences as shown in SEQ ID NO:5 and SEQ ID NO:6; or (6) A nucleotide sequence obtained by modifying, substituting, deleting, or adding one or more bases to the nucleotide sequence described in (5); or (7) A sequence having at least 80% homology to the nucleotide sequence described in (5) or (6); or (8) The complementary sequence of the nucleotide sequence described in (5), (6) or (7).

9. The use of the MDCK cells as described in claim 6 or 7 in the preparation of products for the treatment and / or prevention of influenza viruses.

10. The product, characterized in that, include: The MDCK cells as described in claim 6 or 7.

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