CHO cell strain with complete deletion of glutamine synthetase as well as preparation method and application of CHO cell strain

Abstract

The invention belongs to the field of biosynthesis, and particularly relates to a CHO K1 (ECACC) cell strain with completely inactivated glutamine synthetase (Glul) gene functions as well as a preparation method and application of the CHO K1 (ECACC) cell strain. According to the invention, a base editor CBE4max-SpRY is adopted to target a Glul gene coding region, and a specific codon is efficiently and accurately mutated into a termination codon (TAG / TAA / TGA) through cytosine base directional conversion (C.G-T.A) guided by guide RNA (gRNA) under the condition of not introducing DNA double-strand breakage (DSB), so that complete knockout of the Glul gene is realized. The yield of stably transfected foreign protein of the obtained cell strain is obviously higher than that of commercial CHOZN and ECACC CHO K1 cell lines, and the GS06-10 clone expression quantity of a targeted exon 6 is improved by 3 times compared with that of an ECACC CHO K1 wild type.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biosynthesis, and particularly relates to a CHO cell strain completely lacking glutamine synthetase and a preparation method and application thereof. BACKGROUND

[0002] CHO original cell line is proline-deficient, and needs to add extra proline in the culture medium to grow normally. All known CHO cell lines currently retain this characteristic. CHO original cell line is circulated in different laboratories and companies, and after being cultured, domesticated, modified and re-cloned by different scientists, different types of CHO cell lines are formed. The CHO cell lines we commonly use at present include: ATCC CHO-K1 (wild type), ECACC CHO-K1 (wild type), CHO K1SV (wild type), CHOK1SV GS-KO (GS-deficient), CHOZN CHO K1 (wild type), CHOZN GS (GS-deficient), etc. CHO-K1 is subcloned from the original CHO cell line by Puck laboratory in 1970 and stored in the ATCC (CCL-61) series.

[0003] CHO-K1 was subcloned by ATCC in 1985 and stored in ECACC (85051005). At this time, the cell line began to be used for expression of pharmaceutical or recombinant proteins. The culture method of CHO-K1 cell line is adherent culture, and serum needs to be added in the culture medium to ensure good cell growth. Lonza company domesticated ECACC CHO-K1 through years of modification and established CHO K1SV (Lonza, 2002) cell strain which can adapt to serum-free medium suspension culture and is widely used in GS expression platform. In 2012, CHO cell GS double allele knockout CHOK1SV GS-KO cell strain was launched, which further improved the screening efficiency, shortened the development cycle of stable cell strain, and enhanced the clonal production stability of cells. Merck company obtained CHO-K1 cell strain through ECACC in 2006, domesticated it to a chemically defined medium, and then subcloned to establish CHOZN CHO K1 (Merck, 2006) cell strain. On the basis of this cell line, Merck company knocked out the GS double alleles by ZFN (zinc finger nuclease) technology to obtain GS-deficient cell strain CHOZN GS, and launched it to the market in 2012. At present, multiple projects using CHOZN GS as host cell have been promoted to the clinical experiment stage in many countries around the world.

[0004] CHO cells are the most commonly used mammalian cell culture system. Compared with other systems, CHO cell protein expression has the following advantages: 1. CHO cells allow for precise processing and modification of proteins, making the expressed proteins' biological activity closer to that of natural proteins. 2. CHO cells have strong resistance to shear stress and osmotic pressure, and can be cultured in either adherence or suspension. 3. Exogenous genes integrated into CHO cells can be stably and efficiently amplified and expressed. 4. The expression level of endogenous proteins in CHO cells is low, and the target protein is secreted extracellularly, which is beneficial for the purification of the target protein.

[0005] Glutamine is an essential amino acid for cell growth. Glutamine synthase (GS) catalyzes the synthesis of glutamine from glutamate and ammonia, which is the only way for cells to produce glutamine endogenously. Glutamine serves as an energy source, a component of protein synthesis, and a nitrogen source for the synthesis of purines and pyrimidines in cellular metabolism. Cells lacking GS cannot synthesize glutamine normally and must obtain it from the external culture medium to survive. In the construction of CHO cell lines for biopharmaceuticals, GS is commonly used as a selection marker. Expression vectors containing the target gene and the GS gene are transfected into host cells. By adding the GS inhibitor L-methionine sulfoxide imine (MSX), the enzymatic activity of endogenous GS in the cells can be inhibited. Cells need to increase GS amplification to survive, which also increases the amplification of the target gene, thereby increasing the expression level of the cell line. However, MSX is toxic; its use inhibits cell growth and prolongs the cell line construction cycle. In conventional CHO cell line production, MSX is used during seed amplification. MSX is removed after N-1 or N-2 passages before inoculation, and residual MSX levels are measured during stock culture characterization. As can be seen, the use of MSX increases drug development time and cost. Therefore, if endogenous GS expression in CHO cells can be reduced or completely eliminated, the cell line construction cycle for protein drug development can be accelerated, reducing production costs. Summary of the Invention

[0006] The purpose of this invention is to develop a CHO cell line that can significantly enhance the expression of exogenous proteins. To achieve the above objective, this invention provides the following technical solution: This invention is the first to apply the CBE4max-SpRY system to efficiently knock out the Glul gene in CHO cells. Its core innovations are: 1) Establishing efficient knockout strategies for each of the six exons of the Glul gene, successfully obtaining chassis cell lines with complete deletion of the Glul transcript; 2) Completely avoiding DSB generation, eliminating the risks of DSB-mediated apoptosis, cell cycle arrest, or p53 pathway activation; 3) No resistance genes were introduced.

[0007] The first aspect of this invention discloses a method for preparing a CHO cell line completely lacking glutamine synthase, the method comprising: Using a single-base editor, sgRNA targeting sequences were designed for the CDS sequences of exons 1-6 of the glutamine synthase encoding gene. The designed sgRNA targeting sequences were used to edit the glutamine synthase encoding gene to obtain a CHO cell line completely lacking glutamine synthase. The nucleic acid sequences of the sgRNA include those shown in SEQ ID NO. 7-12.

[0008] The sgRNA sequence targeting the CDS sequence on exon 1 of the glutamine synthase encoding gene is shown in SEQ ID NO: 7; The sgRNA sequence targeting the CDS sequence on exon 2 of the glutamine synthase encoding gene is shown in SEQ ID NO: 8; The sgRNA sequence targeting the CDS sequence on exon 3 of the glutamine synthase encoding gene is shown in SEQ ID NO: 9; The sgRNA sequence targeting the CDS sequence on exon 4 of the glutamine synthase encoding gene is shown in SEQ ID NO: 10; The sgRNA sequence targeting the CDS sequence on exon 5 of the glutamine synthase encoding gene is shown in SEQ ID NO: 11; The sgRNA sequence targeting the CDS sequence on exon 6 of the glutamine synthase encoding gene is shown in SEQ ID NO: 12.

[0009] Preferably, sgRNA sequences are designed using a single-base editor targeting the CDS sequence on exon 6 of the glutamine synthase encoding gene, and the designed sgRNA sequences are used to target and edit the glutamine synthase encoding gene to obtain a CHO cell line completely lacking glutamine synthase.

[0010] As an embodiment of the present invention, the recombinant editing of the exon region of the glutamine synthase encoding gene further includes: CHO cells with knockout positivity were screened for monoclonalization to obtain monoclonal CHO cell lines. Among the monoclonal CHO cell lines, CHO cell lines with complete absence of glutamine synthase were screened.

[0011] Specifically, screening for glutamine synthase-deficient CHO cell lines in monoclonal CHO cell lines includes: Genomic DNA was obtained from monoclonal CHO cell lines, and CHO cell lines completely lacking glutamine synthase were screened using the sequencing data of the genomic DNA.

[0012] The second aspect of the present invention discloses a CHO cell line completely lacking glutamine synthase, wherein the CHO cell line completely lacking glutamine synthase is obtained by the preparation method described above.

[0013] A third aspect of this invention discloses the use of the CHO cell line completely lacking glutamine synthase in expressing a target protein or in preparing engineered cells that express the target protein; the target protein includes an antibody.

[0014] Preferably, the nucleic acid molecule encoding the target protein is introduced into a CHO cell line that is completely deficient in glutamine synthase; and the cell line is cultured under conditions suitable for expressing the target protein, thereby expressing the target protein in the glutamine synthase-deficient CHO cell line.

[0015] The methods for importing the substance include: using a transfection reagent or electrotransfection.

[0016] This invention utilizes a base editor to design targeted sgRNAs to mutate the coding codons in exons 1-6 of the ECACC CHO K1 host cell line to stop codons, thereby knocking out the glutamine synthase gene. Compared to traditional gene editing technologies such as CRISPR / Cas9 and ZFN, this invention has the following significant technical advantages: 1. This invention uses the base editor CBE4max-SpRY to target the coding region of the Gul gene. Without introducing DNA double-strand breaks (DSBs), it efficiently and precisely mutates specific codons into stop codons (TAG / TAA / TGA) through cytosine base direction switching (C·G→T·A) guided by guide RNA (gRNA), achieving complete knockout of the Gul gene. This results in a more efficient and controllable gene knockout effect.

[0017] 2. Completely avoids the generation of DNA double-strand breaks (DSBs), eliminating the risk of cytotoxicity caused by DSBs (such as apoptosis and cell cycle arrest); significantly reduces the risk of large-scale genomic structural instability events such as chromatin translocation caused by DSB error repair.

[0018] 3. This invention knocks out the GS gene in ECACC CHO K1 cells without introducing any resistance genes. During the Gul gene knockout process, guide RNAs were designed to knock out different exons in the Gul gene. The inventors unexpectedly discovered that the monoclonal cell lines obtained after gene editing of different exons showed significant differences in their ability to express exogenous proteins. Among them, the GS06-10 clone obtained by gene editing of the exon 9 region showed a 3-fold increase in stable protein expression compared to ECACC CHO K1. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or existing methods and experiments, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0020] Figure 1 This is a flowchart illustrating the technical solution of the present invention.

[0021] Figure 2 This is a statistical graph showing codon mutations caused by cSNP / Indel in the cell line of Example 5 of the present invention.

[0022] Note: frameshift substitution; nonframeshift substitution; nonsynonymous SNV; stopgain; stoploss; synonymous SNV; unknown.

[0023] Figure 3 This serves as a verification of the absence of blast fungicide resistance, CBE4Max, and sgRNA sequence integration in the GS-KO monoclonal cell line in Example 4 of this invention.

[0024] Note: Among them Figure 3 In the image above, 'a' shows the electrophoresis diagram of the CBE4Max PCR amplification products of cells after 48 hours of transfection and sorting. Figure 3 Image b shows the electrophoresis diagram of the genomic sgRNA-BSD PCR amplification products of cells 48 hours after transfection and sorting. Figure 3 In the middle, c is the electrophoresis diagram of the CBE4Max PCR amplification product of a single clone cell line; Figure 3 Image d in the image is an electrophoresis diagram of the sgRNA-BSD PCR amplification product of a single clone cell line.

[0025] Figure 4 This is a transcriptome differential analysis of the monoclonal cell lines in Example 5 of the present invention.

[0026] Figure 5 The abundance of the GS-KO monoclonal glutamine synthase gene transcript in Example 6 of this invention.

[0027] Figure 6A -B represents the growth curves and viability change curves of different cell batches in Example 7 of this invention.

[0028] Figure 7 This is a graph showing the density of live FB cells in Example 8 of the present invention.

[0029] Figure 8 This is a graph showing the viability of FB cells in Example 8 of the present invention.

[0030] Figure 9 This is a metabolic curve of FB Glc in Example 8 of the present invention.

[0031] Figure 10 This is a graph of FB lactate metabolism in Example 8 of the present invention.

[0032] Figure 11 This is a graph showing the expression level of FB and the relative production rate in Embodiment 8 of the present invention.

[0033] Figure 12 This is a flow cytometry diagram of cell cycle characterization used in Example 10 of the present invention.

[0034] Figure 13 This is a statistical diagram of the cell cycle in each group in Example 10 of the present invention. Detailed Implementation

[0035] The present invention is further illustrated below by way of embodiments, but these embodiments are not intended to limit the invention to their scope. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0036] This invention designs and discloses a method for preparing a CHO cell line completely lacking glutamine synthase, comprising: designing sgRNA targeting sequences for the CDS sequences of exons 1-6 of the glutamine synthase encoding gene using a single-base editor; and using the designed sgRNA targeting sequences to edit the glutamine synthase encoding gene to obtain a CHO cell line completely lacking glutamine synthase; the specific experimental procedure is as follows: Figure 1 As shown.

[0037] Example 1: Construction of gene editing vector The GS gene in CHO cells consists of six exons. GS is primarily responsible for synthesizing glutamine from glutamate and ammonium ions within the cell. Glutamine plays important functions in CHO cells, including providing energy, promoting protein synthesis, regulating lactate metabolism, and acting as an antioxidant and immunomodulatory agent. These functions collectively support the growth and metabolic activities of CHO cells. The partial sequences of exons 1-6 of the GS gene are shown below: GS gene exon 1: AGCACCTTCCACCATGGCCACCTCAGCAAGTTCCCACTTGAACAAAAACATCAAGCAAATGTACTTGTGCCTGCCCCAGGGTGAGAAAGTCCAAGCCATGTATATCTGGGTTGATGGTACTGGAGAAGGACTGCGCTGCAAAACCCGCACCCTGGACTGTGAGCCCAAGTGTGTAGAAG (SEQ ID NO: 1).

[0038] Exon 2 of GS gene: AGTTACCTGAGTGGAATTTTGATGGCTCTAGTACCTTTCAGTCTGAGGGCTCCAACAGTGACATGTATCTCAGCCCTGTTGCCATGTTTCGGGACCCCTTCCGCAGAGATCCCAACAAGCTGGTGTTCTGTGAAGTTTTCAAGTACAACCGGAAGCCTGCAG (SEQ ID NO: 2).

[0039] Exon 3 of GS gene: AGACCAATTTAAGGCACTCGTGTAAACGGATAATGGACATGGTGAGCAACCAGCACCCCTGGTTTGGAATGGAACAGGAGTATACTCTGATGGGAACAGATGGGCACCCTTTTGGTTGGCCTTCCAATGGCTTTCCTGGGCCCCAAG (SEQ ID NO: 3).

[0040] Exon 4 of GS gene: GTCCGTATTACTGTGGTGTGGGCGCAGACAAAGCCTATGGCAGGGATATCGTGGAGGCTCACTACCGCGCCTGCTTGTATGCTGGGGTCAAGATTACAGGAACAAATGCTGAGGTCATGCCTGCCCAG (SEQ ID NO: 4).

[0041] Exon 5 of GS gene: TGGGAATTCCAAATAGGACCCTGTGAAGGAATCCGCATGGGAGATCATCTCTGGGTGGCCCGTTTCATCTTGCATCGAGTATGTGAAGACTTTGGGGTAATAGCAACCTTTGACCCCAAGCCCATTCCTGGGAACTGGAATGGTGCAGGCTGCCATACCAACTTTAGCACCAAGGCCATGCGGGAGGAGAATGGTCTGAA (SEQ ID NO: 5).

[0042] Exon 6 of GS gene:

[0043] (1) Based on the design principle of the targeted guideRNA of the CBE4max-SpRY single base editor, sgRNA targeting sequences were designed for the CDS sequences on exons 1-6 of the GS gene. The sequences are as follows: The sgRNA sequence targeting exon 1 of the GS gene is: 5'-AGTCCAAGCCATGTATATCT-3' (SEQ ID NO: 7).

[0044] The sgRNA sequence targeting exon 2 of the GS gene is: 5'-AGAGTTACCTGAGTGGAATT-3' (SEQ ID NO: 8).

[0045] The sgRNA sequence targeting exon 3 of the GS gene is: 5'-GGAACAGGAGTATACTCTGA-3' (SEQ ID NO: 9).

[0046] The sgRNA sequence targeting exon 4 of the GS gene is: 5'-CCTGCCCAGGTAAATGGCAC-3' (SEQ ID NO: 10).

[0047] The sgRNA sequence targeting exon 5 of the GS gene is: 5'-TTCCAAATAGGACCCTGTGA-3' (SEQ ID NO: 11).

[0048] The sgRNA sequence targeting exon 6 of the GS gene is: 5'-ATTCGAGCCTACGATCCCAA-3' (SEQ ID NO: 12).

[0049] (2) Synthesize sgRNA sequence primer pairs (Shanghai Platinum Biotechnology Co., Ltd.), anneal the primer pairs to obtain the sgRNA insert fragment. The annealing system is as follows: 10 μL Oligo Primer F (10 μM), 10 μL Oligo Primer R (10 μM). The annealing program is as follows: 98℃ for 5 min, then gradually decrease to 37℃ at a rate of 0.1℃ / s, and 37℃ for 5 min.

[0050] The sequences of the forward and reverse oligonucleotide chains targeting exon 1 of the GS gene are shown below: Forward oligonucleotide chain (Oligo Primer F): 5'-ACCGAGTCCAAGCCATGTATATCT-3' (SEQ ID NO: 13); Reverse oligonucleotide chain (Oligo Primer R): 5'-AAACAGATATACATGGCTTGGACT-3' (SEQ ID NO: 14).

[0051] The sequences of the forward and reverse oligonucleotide chains targeting exon 2 of the GS gene are shown below: Forward oligonucleotide chain (Oligo Primer F): 5'-ACCGAATTCCACTCAGGTAACTCT-3' (SEQ ID NO: 15); Reverse oligonucleotide chain (Oligo Primer R): 5'-AAACAGAGTTACCTGAGTGGAATT-3' (SEQ ID NO: 16).

[0052] The sequences of the forward and reverse oligonucleotide chains targeting exon 3 of the GS gene are shown below: Forward oligonucleotide chain (Oligo Primer F): 5'-ACCGGGAACAGGAGTATACTCTGA-3' (SEQ ID NO: 17); Reverse oligonucleotide chain (Oligo Primer R): 5'-AAACTCAGAGTATACTCCTGTTCC-3' (SEQ ID NO: 18).

[0053] The sequences of the forward and reverse oligonucleotide chains targeting exon 4 of the GS gene are shown below: Forward oligonucleotide chain (Oligo Primer F): 5'-ACCGCCTGCCCAGGTAAATGGCAC-3' (SEQ ID NO: 19); Reverse oligonucleotide chain (Oligo Primer R): 5'-AAACGTGCCATTTACCTGGGCAGG-3' (SEQ ID NO: 20).

[0054] The sequences of the forward and reverse oligonucleotide chains targeting exon 5 of the GS gene are shown below: Forward oligonucleotide chain (Oligo Primer F): 5'-ACCGCCATTCCAGTTCCCAGGAAT-3' (SEQ ID NO: 21); Reverse oligonucleotide chain (Oligo Primer R): 5'-AAACATTCCTGGGAACTGGAATGG-3' (SEQ ID NO: 22).

[0055] The sequences of the forward and reverse oligonucleotide chains targeting exon 6 of the GS gene are shown below: Forward oligonucleotide chain (Oligo Primer F): 5'-ACCGATTCGAGCCTACGATCCCAA-3' (SEQ ID NO: 23); Reverse oligonucleotide chain (Oligo Primer R): 5'-AAACTTGGGATCGTAGGCTCGAAT-3' (SEQ ID NO: 24).

[0056] (3) The PGL3-U6-ACCG-BSD vector was digested with the endonuclease BsaI and then recovered by gel excision.

[0057] (4) The double-stranded sgRNA obtained from annealing in step (2) was ligated into the PGL3-U6-ACCG-BSD vector. The ligation system was as follows: 1 μL PGL3-U6-ACCG-BSD-BsaI digestion gel recovery (50 ng), 1 μL annealed double-stranded sgRNA fragment, 0.5 μL 10×T4 ligation buffer (Takara), 0.5 μL T4 ligase (Takara), and 2 μL sterile distilled water. Ligation was carried out at 16℃ for 1 h. Then, the ligation product was transformed. 50 μL of E. coli DH5α was added to the product, and after heat shock at 42℃, it was immediately placed on ice. The product was then plated onto LB agar plates containing ammonium phosphate and incubated at 37℃ for 13 h. After picking single-clone colonies and culturing them in a shaker, plasmids were extracted and sequenced to obtain the gene editing vectors PGL3-U6-ACCG-BSD-GS01 (sgRNA targeting exon 1 of the GS gene), PGL3-U6-ACCG-BSD-GS02 (sgRNA targeting exon 2 of the GS gene), PGL3-U6-ACCG-BSD-GS03 (sgRNA targeting exon 3 of the GS gene), PGL3-U6-ACCG-BSD-GS04 (sgRNA targeting exon 4 of the GS gene), PGL3-U6-ACCG-BSD-GS05 (sgRNA targeting exon 5 of the GS gene), and PGL3-U6-ACCG-BSD-GS06 (sgRNA targeting exon 6 of the GS gene).

[0058] Example 2: Precise single-base editing targeting the GS gene in CHO-K1 cells and identification of monoclonal cells (1) Plasmid extraction: a) Receiving plasmid HY-103 / PGL3-U6-ACCG-BSD-GS01; HY-103 / PGL3-U6-ACCG-BSD-GS02; HY-103 / PGL3-U6-ACCG-BSD-GS03; HY-103 / PGL3-U6-ACCG-BSD-GS04; HY-103 / PGL3-U6-ACCG-BSD-GS05; YP1068 / PGL3-U6-ACCG-BSD-GS06.

[0059] b) Take competent cells (E.coli DH5α Competent Cells), rinse them gently with running water until they are 1 / 3 or 1 / 2 thawed, and place them in an ice box. Add an appropriate amount of plasmid to the competent cells (not exceeding 1 / 10 of the competent cell volume) and place them in an ice box for 5 minutes.

[0060] c) Heat shock at 42℃ for 45 seconds.

[0061] d) Transfer competent cells to an ice box and place for 2 minutes.

[0062] e) Add to LB medium containing the corresponding resistance and incubate at 220 rpm and 37°C for 12-16 hours with shaking.

[0063] f) Centrifuge at 6000 rpm, 4℃ for 7 min to collect the bacterial cells, then invert the bottle to drain the water.

[0064] g) Add 10 mL of Buffer RES-EF, resuspend the bacterial pellet, and transfer it to a 50 mL centrifuge tube.

[0065] h) Add 10.5 mL of Buffer LYS-EF, gently and thoroughly mix by turning the container upside down to ensure complete lysis of the bacteria until a clear solution is formed.

[0066] i) Prepare the column and slowly add 15 mL of Buffer EQU-EF to the edge of the filter membrane to equilibrate the column.

[0067] j) Add 11 mL of Buffer NEU-EF, gently and thoroughly invert the centrifuge tube 6-10 times to mix, until the liquid becomes colorless.

[0068] k) 4000g, centrifuged at 4℃ for 10 minutes, and the supernatant was collected.

[0069] l) Pour the supernatant after centrifugation into a collection column with a filter membrane.

[0070] m) First wash: Add 5 mL of Buffer FIL-EF along the edge of the filter membrane.

[0071] n) Second wash: Discard the filter membrane and add 45 mL of Buffer ENDO-EF.

[0072] o) Third wash: Add 25 mL Buffer WASH-EF.

[0073] p) Add 5 mL of Buffer ELU-EF to elute the DNA.

[0074] q) Add 3.5 mL of isopropanol to the eluted DNA to precipitate the DNA, and incubate at room temperature for 2 min.

[0075] r) Pour the liquid into the Finalizer, add 2 mL of 70% EtOH, and purge air more than 6 times to dry the residual ethanol.

[0076] s) Dry the finalizer and collect the DNA using 500-1000 μL H2O-EF. Measure the plasmid concentration.

[0077] t) Adjust the plasmid concentration to approximately 1000 ng / μL for transfection.

[0078] (2) Cell culture: a) Resuscitate ECACC CHO K1 cells and passage them using CD CHO Fusion + 6mM L-Glutamine. Passage should be performed every 2-3 days at a density of (0.2-0.4) E6 cells / ml. The cell doubling time is approximately 16-20 hours. Shake culture conditions are 8% CO2, 37℃, 120 rpm, and 80% humidity.

[0079] b) Passage at 0.5 E6 Cells / ml 24 hours before transfection.

[0080] (3) Cell transfection: a) Cell counting: Mix 20 μL of cell suspension with 20 μL of trypan blue and count the cells using Countstar.

[0081] b) T-bottle preparation: Prepare two T-bottles for each transfection group, add 19ml CD CHO Fusion + 6mM L-Glutamine for later use.

[0082] c) Host cell centrifugation: Based on the counting results, calculate and take the required suspension volume of 1E7 cells into a 50ml centrifuge tube, centrifuge at 800rpm for 5min, and discard the supernatant.

[0083] d) The transfection ratio was 1E7 Cells + 15 μg plasmid (with an editor to SgRNA ratio of 3:1). The electroporation buffer was CD CHO Fusion + 6 mM L-Glutamine (pre-cooled). A Bio-Rad electroporator was used with parameters set to 300 V and 950 μF. Every two electroporations were combined, and the electroporated cells were transferred into the T-flask described in (b). Samples were taken after 48 h to detect the knockout efficiency.

[0084] e) Perform monoclonal plating based on transfection efficiency.

[0085] (4) Monoclonal plate laying: a) 48 hours after transfection, add blast fungicide at a final concentration of 1 μg / ml for pesticide screening.

[0086] b) After 3 days of pressure culture with blast fungicide, collect the cell suspension and centrifuge. Discard the supernatant after centrifugation and add fresh medium CD CHO Fusion + 6mM L-Glutamine. After the cells recover (viability > 90%), perform monoclonal plating.

[0087] c) Limiting dilution: Take cells and perform serial dilution, gradually diluting from E6 Cells / ml to E3 Cells / ml.

[0088] d) Preparation of cloning medium: EX-CELL CHO CloningMedium containing 6 mM L-Glutamine.

[0089] e) Plate the cells at a rate of 1 Cell / well / 200μL. After plating, place the cells in a CO2 incubator and culture them at 37°C with 8% CO2 and a water tray for humidification.

[0090] f) After approximately 20 days of culture in well plates, cells with a confluence greater than 30% are transferred to 24WP culture, and after 3-4 days, transferred to 6WP culture. Once the cells have recovered, samples are taken for sequencing. The selection medium for the well plates is CD CHO Fusion + 6mM L-Glutamine.

[0091] (5) Large-scale culture of monoclonal cells: a) Positive clones confirmed by sequencing were transferred to SF125 and cultured in a shaker at 37°C, 8% CO2, and 80% humidity. The subculture medium used was the same as that used for plate screening.

[0092] b) After cell viability was restored, library construction was performed at 1E7 cells / vial. The cryopreservation medium was CD CHO Fusion:DMSO = 9:1.

[0093] Example 3: Determination of the GS gene base sequence in CHO-K1 cells edited by CBE4Max (1) The CDS fragment of the GS gene from the monoclonal cell line amplified in Example 2 was targeted and sequenced. a) Use the One Step Mouse Genotyping Kit (Vazyme) to extract the genome of a single-clone cell line according to the instructions.

[0094] b) Using the genome of a single cloned cell line as a template, the CDS sequences of exons 1-6 of the GS gene were amplified by PCR. The primer sequences and PCR reaction systems are shown below: GS01 forward primer (GS01F): 5'-CCAACCTACAAGGGATGATC-3' (SEQ ID NO: 25); GS01 reverse primer (GS01R): 5'-TCTATGCCTCCAGACTACTC-3' (SEQ ID NO: 26); GS02 forward primer (GS02F): 5'-TCCCAGGAACTGAAGTTAAC-3' (SEQ ID NO: 27); GS02 reverse primer (GS02R): 5'-GTTTTTCAGCAGGCAAGTTC-3' (SEQ ID NO: 28); GS03 forward primer (GS03F): 5'-TTTGCTCTCACTCCATTCCC-3' (SEQ ID NO: 29); GS03 reverse primer (GS03R): 5'-TCTCATCACCATCCTGTCAC-3' (SEQ ID NO: 30); GS04 forward primer (GS04F): 5'-GCAAGTCTTAGGACCTTGTC-3' (SEQ ID NO: 31); GS04 reverse primer (GS04R): 5'-GGGTCCTATTTGGAATTCCC-3' (SEQ ID NO: 32); GS05 forward primer (GS05F): 5'-GCAAGTCTTAGGACCTTGTC-3' (SEQ ID NO: 33); GS05 reverse primer (GS05R): 5'-GGGTCCTATTTGGAATTCCC-3' (SEQ ID NO: 34); GS06 forward primer (GS06F): 5'-CAGGAACAAATGCTGAGGTC-3' (SEQ ID NO: 35); GS06 reverse primer (GS06R): 5'-TAACACAAAGCCCTTCCACC-3' (SEQ ID NO: 36); The PCR reaction system is shown below: 50μL reaction system: Taking GS01 cells as an example, 500ng of monoclonal cell template genomic DNA, 2μL of primer GS01F with a concentration of 10μm, 2μL of primer GS04R with a concentration of 10μm, 25μL of 2×phanta buffer, and add sterile distilled water to a total volume of 50μL. The genomic CDS sequence of GS01 monoclonal CHO cells can be amplified to a 524bp fragment.

[0095] The PCR program was standardized as follows: 98℃ for 5 min; 95℃ for 30 s, 56℃ for 30 s, 72℃ for 30 s, 35 cycles; 72℃ for 5 min; 4℃ ∞.

[0096] (2) The PCR amplification products of exons 1 to 6 of the GS gene were recovered by agarose gel electrophoresis and sent to the company (Shanghai Platinum Biotechnology Co., Ltd.) for DNA fragment sequencing using primers GS01 to 06F / R.

[0097] (3) Sequencing results showed that GS01-07, GS02-07, GS03-05, GS04-142, GS05-10 and GS06-10 underwent base substitution at the sgRNA recognition binding site to form a stop codon. After editing, the translation of the GS gene was terminated prematurely. The CDS sequence after editing is shown in SEQ ID NO: 44. The sequencing map peaks were clear and no double peaks were observed at the base substitution sites. Taking GS01-07 clone as an example, the GS gene of the single clone cell was translated into the stop codon TAA at 27Q in exon 1. A nonsense mutation was generated on the CDS sequence and was recognized as the stop codon X (UAA) during transcription and translation. After the mutation, the translation was terminated prematurely, achieving the GS gene knockout effect.

[0098] 1. Edited sequence of exon 1 of monoclonal GS gene (GS01-07) (cytosine to thymine substitution at 27Q): ATGGCCACCTCAGCAAGTTCCCACTTGAACAAAAACATCAAGCAAATGTACTTGTGCCTGCCCCAGGGTGAGAAAGTCCAAGCCATGTATATCTGGGTTGATGGTACTGGAGAAGGACTGCGCTGCAAAACCCGCACCCTGGACTGTGAGCCCAAGTGTGTAGA (SEQ ID NO: 37).

[0099] 2. Edited sequence of exon 2 of monoclonal GS gene (GS02-07) (60W guanine to adenine replacement): AGAGTTACCTGAGTGGAATTTTGATGGCTCTAGTACCTTTCAGTCTGAGGGCTCCAACAGTGACATGTATCTCAGCCCTGTTGCCATGTTTCGGGACCCCTTCCGCAGAGATCCCAACAAGCTGGTGTTCTGTGAAGTTTTCAAGTACAACCGGAAGCCTGCAG (SEQ ID NO: 38).

[0100] 3. Edited sequence of exon 3 of the GS gene from monoclonal GS03-05 (replacement of cytosine to thymine at 135Q): AGACCAATTTAAGGCACTCGTGTAAACGGATAATGGACATGGTGAGCAACCAGCACCCCTGGTTTGGAATGGAACAGGAGTATACTCTGATGGGAACAGATGGGCACCCTTTTGGTTGGCCTTCCAATGGCTTTCCTGGGCCCCAAG (SEQ ID NO: 39).

[0101] 4. Edited sequence of exon 4 of monoclonal GS gene (GS04-142, cytosine to thymine substitution in 201Q): GTCCGTATTACTGTGGTGTGGGCGCAGACAAAGCCTATGGCAGGGATATCGTGGAGGCTCACTACCGCGCCTGCTTGTATGCTGGGGTCAAGATTACAGGAACAAATGCTGAGGTCATGCCTGCCCAG (SEQ ID NO: 40).

[0102] 5. Edited sequence of exon 5 of monoclonal GS gene (GS05-010) (cytosine to thymine replacement at 205Q): TGGGAATTCCAAATAGGACCCTGTGAAGGAATCCGCATGGGAGATCATCTCTGGGTGGCCCGTTTCATCTTGCATCGAGTATGTGAAGACTTTGGGGTAATAGCAACCTTTGACCCCAAGCCCATTCCTGGGAACTGGAATGGTGCAGGCTGCCATACCAACTTTAGCACCAAGGCCATGCGGGAGGAGAATGGTCTGAA (SEQ ID NO: 41).

[0103] 6. Edited sequence of exon 6 of the GS gene from monoclonal GS06-10 (cytosine to thymine replacement in 286R): GCACATCGAGGAGGCCATCGAGAAAACTAAGCAAGCGGCACCGGTACCACATTCGAGCCTACGATCCCAAGGGGGGCCTGGACAATGCCCGTCGTCTGACTGGGTTCCACGAAACGTCCAACATCAACGACTTTTCTGCTGGTGTCGCCAATCGCAGTGCCA GCATCCGCATTCCCCGGACTGTCGGCCAGGAGAAGAAAGGTTACTTTGAAGACCGCCGCCCCTCTGCCAATTGTGACCCCTTTGCAGTGACAGAAGCCATCGTCCGCACATGCCTTTCCAATGAGACTGGCGACGAGCCCTTCCAATACAAAAACTAA (SEQ ID NO: 42).

[0104] Example 4: Verification of monoclonal cell lines lacking resistance to blast fungicide (BSD), CBE4Max, and sgRNA sequence integration. (1) The genomes of the monoclonal cell lines obtained in Example 2 were extracted using the One Step Mouse Genotyping Kit (Vazyme) according to the method in the instruction manual.

[0105] (2) Using the genomes of transfected cell populations and monoclonal cell lines as templates, CBE4Max, sgRNA, and internal fragments of the blast fungicide resistance gene were amplified by PCR to detect whether the CBE4Max, blast fungicide resistance gene, and sgRNA gene were integrated into the genome of GS-deficient monoclonal cell lines. The primer pair sequences are as follows: PCR CBE4Max F: 5'-TGAAGTGTTCTTTGACCCCC-3' (SEQ ID NO: 43); PCR CBE4Max R: 5'-TGATCTCGGTGTTCACTCTC-3' (SEQ ID NO: 44); PCR sgRNA-BSD F: 5'-TGAAAAGTGGCACCGAGTC-3' (SEQ ID NO: 45); PCR sgRNA-BSD R: 5'-TGAAGAAGTCGTGCTGCTTC-3' (SEQ ID NO: 46); 50μL reaction system: Taking GS01 cells as an example, 500ng of monoclonal cell template genomic DNA, 2μL of primer GS01F with a concentration of 10μm, 2μL of primer GS04R with a concentration of 10μm, 25μL of 2×phanta buffer, and add sterile distilled water to a total volume of 50μL.

[0106] The PCR program was standardized as follows: 98℃ for 5 min; 95℃ for 30 s, 56℃ for 30 s, 72℃ for 30 s, 35 cycles; 72℃ for 5 min; 4℃ ∞.

[0107] (3) By observing whether there is a target band by agarose gel electrophoresis, it can be determined whether the genome of the monoclonal cell line has the rice blast fungicide resistance gene, CBE4Max and sgRNA sequence integration.

[0108] The results are as follows Figure 3 As shown, this method was used to obtain the integration of the blast fungicide resistance gene, CBE4Max, and sgRNA sequence into a single clonal cell line.

[0109] Example 5: Differential Transcriptome Analysis of Monoclonal Cell Lines (1) Sample processing and sequencing outsourcing Gene-edited monoclonal cell lines (ATCC-CHO-K1, CHOZN, GS 06-10) and wild-type cell line (ECACC-CHO-K1) were collected and sent for testing in three groups (experimental and control). Total RNA was extracted using the TRIzol method. After passing Agilent 2100 quality control (RIN≥8.0), the following services were provided by Yunzhun Medical Technology Co., Ltd.: a) Sequencing on the Illumina NovaSeq 6000 platform (PE150) b) Standard bioinformatics analysis: including raw data quality control (FastQC), sequence alignment (Hisat2 v2.2.1), and gene quantification (featureCounts v2.0.3). (2) Analysis of codon mutations caused by cSNP / Indel a) Data processing workflow using the R language package Varscan: Key parameter settings: SNP site base Q > 20; The number of reads covering this site is >8; The number of reads supporting the mutation site is >2; The p-value of the SNP site is <0.01.

[0110] This invention statistically analyzed the various types of codon mutations in each sample, and the results are as follows: Figure 2 As shown, the codon mutations caused by cSNP / Indel in the obtained monoclonal cell lines are within the normal range, indicating high safety.

[0111] (3) Differentially expressed gene analysis a) Data processing flow using the R language package DESeq2: Key parameter settings: Standardization method: default median of ratios method Significance threshold: Corrected p-value (padj) < 0.05 Difference multiple threshold: |log2(FoldChange)|>1 (4) Visualization of differentially expressed genes The R language package ggplot was used to visualize differentially regulated genes and generate a volcano plot. In the plot, green dots represent genes that are significantly downregulated compared to the ECACC-CHO-K1 cell line, blue dots represent genes that are significantly upregulated, and gray dots represent non-differentially regulated genes.

[0112] The results are as follows Figure 4 As shown, the glutamine synthase gene (Glul) in the monoclonal cell line GS06-10 obtained by this method was significantly downregulated and expressed at a low level.

[0113] Example 6: Characterization of GS gene (Glul) transcript abundance in GS-deficient monoclonal cell lines lacking integration of blast fungicide resistance gene, CBE4Max, and sgRNA sequences. (1) RT-qPCR primers binding to exons 1, 3, and 6 of the GS gene were designed to detect the degree of glutamine synthase gene deletion at the RNA level in GS-deficient cell lines. The primer pair sequences are as follows: qPCR GS-E1 F: 5'-ATGGCCACCTCAGCAAGTTC-3' (SEQ ID NO: 47); qPCR GS-E1 R: 5'-CGCAGTCCTTCTCCAGTACC-3' (SEQ ID NO: 48); qPCR GS-E3 F: 5'-TTAAGGCACTCGTGTAAACGG-3' (SEQ ID NO: 49); qPCR GS-E3 R: 5'-GAAAGCCATTGGAAGGCCAAC-3' (SEQ ID NO: 50); qPCR GS-E6 F: 5'-CGGCCAGGAGAAGAAAGGTT-3' (SEQ ID NO: 51); qPCR GS-E6 R: 5'-ATTGAGAAGGCATGTGCGGA-3' (SEQ ID NO: 52).

[0114] (2) Take 1×10 from each 6 Wild-type CHO-K1 cells, cell lines GS01-07, GS02-07, GS03-05, GS04-142, GS05-10, and GS06-10, were centrifuged at 1000 rpm for 5 min and the supernatant was discarded. Add 1 mL of Trizol (Invitrogen) to the cell pellet, disperse the homogenized tissue, and shake to mix. Add 200 μL of phenol-chloroform, mix, and let the solution stand at room temperature for 5 min after it turns milky white. Centrifuge at 12000 rpm for 15 min at 4 °C, transfer 300 μL of the clear organic phase to 600 μL of isopropanol, invert to mix, and let stand at room temperature for 15 min. Centrifuge at 12000 rpm for 15 min at 4 °C, discard the supernatant, add 900 μL of 75% ethanol, and vortex. Centrifuge at 12000 rpm for 5 min, discard the supernatant, and centrifuge at 12000 rpm for 5 min. Discard the residual liquid and dry until the DNA pellet becomes clear. Add 30 μL of RNase-free water and dissolve at 75 °C for 5 min.

[0115] cDNA was synthesized by reverse transcription and genomic DNA was removed using the TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix kit for subsequent quantitative real-time PCR experiments.

[0116] The PerfectStart® Green qPCR SuperMix kit was used for quantitative real-time PCR experiments using the fluorescent dye method.

[0117] The PCR program was standardized as follows: 95℃ for 5 min; 95℃ for 30 s, 60℃ for 30 s, 72℃ for 30 s, for 40 cycles. Results are as follows... Figure 5 As shown.

[0118] Example 7: Verification of GS gene functional defects The six knockout positive clones (GS01-07, GS02-07, GS03-05, GS04-142, GS05-10, and GS06-10) and the wild-type ECACC CHO K1 (a total of seven cell lines) were placed in VegaCHO Medium OPM medium containing 0 mM Glutamine / 1 mM Glutamine / 2 mM Glutamine / 6 mM Glutamine. Cell growth and viability data were monitored daily at a seeding density of 0.5 E6 Cells / ml * 30 ml. Cell growth and viability curves are shown in [Figure number missing]. Figure 6A and Figure 6B .

[0119] Experimental results: Of the 7 cell lines, except for ECACC CHO K1 and GS05-10, the others showed the following characteristics: almost no growth in medium containing 0 mM Glutamine, slow growth in medium containing 1 mM Glutamine / 2 mM Glutamine, and high density and viability in medium containing 6 mM Glutamine.

[0120] In conclusion, the successfully gene-edited cell lines GS01-07, GS02-07, GS03-05, GS04-142, and GS06-10 could not grow in glutamine-free medium, indicating that the gene was not functioning properly.

[0121] Example 8: Positive clone stable transformation experiment (1) Six cell lines were revived and passaged in CD CHO Fusion + 6 mM Glutamine medium. After the cells recovered, they were prepared for electroporation.

[0122] (2) The cells were passaged at 0.5E6 Cells / ml one day before transfection and transfected using the pembrolizumab plasmid.

[0123] Transfection was performed using a Bio-Rad electroporator, 1E7 Cells + 40 μg DNA, at 300 V and 950 uF.

[0124] After electroporation, cells were transferred to T-flasks for culture. Transient expression levels were measured after 24 hours, and the expression level was approximately 1 μg / ml. Cells were then collected and transferred to 24WP culture medium and cultured using C. NEST with CD-CHO Fusion medium.

[0125] (3) After culturing for 3 days, the cells in 24WP were transferred to SF125 for culture. The culture conditions on the shaker were 37℃, 70rpm, and 80% humidity.

[0126] (4) After the cells slowly recover, gradually increase the shaking speed from 70 rpm to 90 rpm, and finally to 120 rpm.

[0127] (5) After 6 pool cells have fully recovered, they are seeded into FB at a rate of 0.5E6 Cells / ml * 30ml, using VegaCHO. The feeding regimen is 4% / 5% / 6% / 6% / 5% / 5% VegaCHO Feed and 1 / 10 of CDFS36 on days 3 / 5 / 7 / 9 / 11 / 13. Harvest after 16 days of culture or when the viability is less than 60%.

[0128] (6) VCD and Via data during the FB culture process of the 6 pools are as follows: Figure 7~Figure 8 As shown. After 16 days of incubation, the Peak VCD range was (19.2~21.5)×10. 6 The cell count / ml at harvest was 88.1%–94.61% (Via) and the IVCD range was (189.1–208.0) × 10⁻¹⁰. 6 cells / ml·days. Glucose metabolism level and lactate metabolism level as follows: Figure 9~Figure 10 As shown. Antibody expression levels in FB supernatant were detected using ForteBio, with expression levels ranging from (0.24~0.74) g / L and Qp ranging from (1.3~3.7) pg / cell / day (see details). Figure 11 (and Table 1).

[0129] Table 1. Antibody expression levels and specific production rates Minipool Peak VCD (10 6 cells / ml)]]> Harvest Via (%) Titer (g / L) IVCD (10 6 cells / ml·days) Qp (pg / cell / day) GS01-07 21.5 90.45 0.43 208.0 2.1 GS02-07 19.2 88.1 0.46 189.1 2.4 GS03-05 19.5 91.58 0.55 202.8 2.7 GS06-10 19.6 91.08 0.74 199.6 3.7 GS04-142 21.3 88.99 0.32 196.1 1.6 ECACC CHO K1 19.0 94.61 0.24 189.0 1.3 Example 9: GO enrichment analysis The RNA-SEQ results were analyzed using the topGO toolkit (v2.40.0) in the R language environment (v4.1.0) to perform gene ontology enrichment analysis on differentially expressed genes (DEGs). The specific process is as follows: First, the list of DEGs obtained by screening (screening criteria: |log2FC|>1 and FDR<0.05) was converted into Entrez ID format; then, the org.Crg.eg.db annotation database was called to construct the analysis object, and the ontology type was set as biological process (BP), molecular function (MF) or cellular component (CC), and the patent-optimized node filtering parameter (nodeSize=5 to eliminate interference from low-frequency GO terms) was applied; the top 10 significantly enriched GO entries and their enrichment fold, p-value and other parameters were output. GO enrichment analysis (molecular function (MF), biological process (BP), and cellular component (CC)) showed significant differences in the MF category among the GS09-10 cell lines: The SH3 domain binding term was significantly downregulated, and the mammalian homolog of the term-enriched gene ENSCGRG00001012857 is SLC38A2 (encoding a glutamine transporter). The SLC38A2 protein, SNAT2, belongs to the SLC38 solute carrier family and is a Na⁺-dependent, pH-sensitive, neutral amino acid transporter, with glutamine (Gln) as its main substrate. This result suggests that the E6 cell line has a significantly reduced glutamine transport capacity. Simultaneously, ATP-gated ion channel activity and proton-transporting ATP synthase were also significantly reduced in the MF category. The term "complex" was significantly upregulated, with enriched genes including ENSGRG00001017108 (P2rx6), ENSGRG00001023059 (P2rx4), and ENSGRG00001009513 (Atp5k, encoding ATP synthase subunit k). P2rx6 and P2rx4, as ATP-gated ion channel receptors, activate downstream pathways by mediating increased intracellular Ca²⁺ concentration: In metabolic reprogramming, Ca²⁺ regulates the glycolysis / oxidative phosphorylation balance by activating the AMPK / mTOR pathway and drives HIF-1α stabilization, promoting the Warburg effect; in proliferation regulation, Ca²⁺-dependent kinases (such as CaMKII) activate STAT3 and ERK signaling to promote cell cycle progression (inducing Cyclin...). Atp5k encodes ATP synthase subunit k, a key component of oxidative phosphorylation (OXPHOS) terminal enzyme ATP synthase (Complex V), which catalyzes the ADP+Pi→ATP reaction. Upregulation of its expression leads to an increase in intracellular ATP levels, driving the synthesis of biomolecules such as proteins and nucleic acids, thereby accelerating the cell cycle.This indicates that the cell line obtained by this invention has a high level of metabolic proliferation.

[0130] Example 10: PI staining to characterize the cell cycle (1) Cell treatment and fixation Take 1×10 6 Cells in logarithmic growth phase (cells / mL) were collected, centrifuged at 1000g for 5 min, washed twice with pre-cooled PBS, and fixed at 4℃ for ≥12 h by slowly adding 70% ethanol. After centrifugation at 1000g for 5 min, the ethanol was discarded; staining working solution PI (50 μg / mL) + RNase A (100 μg / mL) + PBS was prepared; cells were resuspended in 500 μL of staining solution and incubated at 37℃ in the dark for 30 min. Flow cytometry analysis: Flow cytometry parameters: Excitation wavelength: 488nm, emission channel: FL2 or FL3 (>575nm long-pass filter), cell count: ≥100,000 events / sample. Threshold settings: FSC / SSC scatter plot to delineate viable cell populations, excluding adhesions (PI-Area vs PI-Width dual-parameter gating), PI-W histogram to characterize cell cycle.

[0131] (2) Data Analysis Cell cycle fitting was performed using Flowjo 10.9 software, with the Dean-Jett-Fox model selected. Statistical results were obtained.

[0132] like Figure 12 , 13 As shown, the GS09-10 group of cells, after quantitative treatment, exhibited significant proliferation and activation characteristics in their cell cycle distribution. Flow cytometry analysis revealed that the proportion of cells in G1 phase decreased to 35%, a 7% decrease compared to ZN (42%), indicating that cells accelerated through the growth preparation phase; the proportion of cells in S phase increased to 35%, a 12% increase compared to ZN (23%), demonstrating significantly enhanced DNA replication activity; and the proportion of cells in G2 phase reached 24%, reflecting that cells that had completed replication efficiently entered the division preparation stage.

[0133] This data confirms that the cell line population obtained by this invention has high proliferation activity.

[0134] In summary, based on continuous passage and batch data of the six cell lines in different concentrations of glutamine medium, it can be concluded that, except for ECACC CHO K1 and GS05-10, the endogenous glutamine produced by the other five cell lines after GS gene knockout is insufficient to sustain their growth, and therefore they can hardly grow in glutamine-free medium. Combined with data on FB expression levels and growth metabolism in the stable transgenic cell lines, GS06-10 was determined to be the optimal clone. This invention successfully obtained five ECACC CHOK1 knockout cell lines, all of which showed improved stable protein expression levels compared to the commercially available knockout cell line ECACC CHO K1. Among them, the GS06-10 clone showed a 3-fold increase in stable protein expression.

[0135] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a CHO cell line completely lacking glutamine synthase, characterized in that, The preparation method includes: Using the single-base editor CBE4Max-SpRY, an sgRNA targeting sequence was designed for the CDS sequence on the exon of the glutamine synthase encoding gene in ECACC CHO K1 cell line. The coding sequence in the exon was mutated to a stop codon, thereby knocking out the glutamine synthase encoding gene and obtaining a CHO cell line completely lacking glutamine synthase. The exon of the glutamine synthase encoding gene is exon 6, and the sgRNA sequence targeting the CDS sequence on exon 6 of the glutamine synthase encoding gene is shown in SEQ ID NO:

12.

2. The method for preparing a CHO cell line completely lacking glutamine synthase according to claim 1, characterized in that, The exons of the glutamine synthase encoding gene also include exons 1-5; The sgRNA sequence targeting the CDS sequence on exon 1 of the glutamine synthase encoding gene is shown in SEQ ID NO: 7; The sgRNA sequence targeting the CDS sequence on exon 2 of the glutamine synthase encoding gene is shown in SEQ ID NO: 8; The sgRNA sequence targeting the CDS sequence on exon 3 of the glutamine synthase encoding gene is shown in SEQ ID NO: 9; The sgRNA sequence targeting the CDS sequence on exon 4 of the glutamine synthase encoding gene is shown in SEQ ID NO: 10; The sgRNA sequence targeting the CDS sequence on exon 5 of the gene encoding glutamine synthase is shown in SEQ ID NO:

11.

3. A method for preparing a CHO cell line completely lacking glutamine synthase according to any one of claims 1-2, characterized in that, Recombinant editing of the exon regions of the glutamine synthase encoding gene also includes: CHO cells with knockout positivity were screened for monoclonalization to obtain monoclonal CHO cell lines, and CHO cell lines with complete absence of the glutamine synthase were screened from the monoclonal CHO cell lines.

4. The method for preparing a CHO cell line completely lacking glutamine synthase according to claim 3, characterized in that, Screening for glutamine synthase-deficient CHO cell lines in monoclonal CHO cell lines specifically includes: Genomic DNA was obtained from monoclonal CHO cell lines, and CHO cell lines completely lacking glutamine synthase were screened using the sequencing data of the genomic DNA.

5. A CHO cell line completely lacking glutamine synthase, characterized in that, The CHO cell line completely lacking glutamine synthase was obtained by the preparation method according to any one of claims 1-4.

6. The application of the CHO cell line completely lacking glutamine synthase as described in claim 5 in expressing a target protein or in preparing engineered cells expressing the target protein; wherein the target protein includes an antibody.

7. The application according to claim 6, characterized in that, The nucleic acid molecule encoding the target protein is introduced into a CHO cell line that is completely deficient in glutamine synthase; the cell line is cultured under conditions suitable for expressing the target protein, and the glutamine synthase-deficient CHO cell line expresses the target protein.

8. The application according to claim 7, characterized in that, The methods for importing the substance include: using transfection reagents or electrotransfection.

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