CN112126661B - Method for efficiently knocking out TIGIT gene in NK cell - Google Patents

Abstract

The invention discloses a method for efficiently knocking out TIGIT gene in NK cells. The method uses CRISPR/Cas9 gene editing system to introduce the complex of sgRNA and Cas9 protein into amplified NK cells by electroporation transfection. wherein: the sequence of the sgRNA is selected from any one of the sequences shown in SEQ ID NO.1-4. The method of the invention can realize the knockout efficiency of TIGIT gene in NK cells close to 80%, not only the knockout efficiency is high, but also the obtained NK cells that do not express TIGIT can relieve the immunosuppressive effect of CD155+ tumor cells on NK cells Compared with wild-type NK cells, it exhibits stronger anti-tumor activity in both in vivo and in vitro experiments, significantly improving the recognition and killing activities of NK cells on tumor cells, and is expected to be developed into a safe and effective anti-tumor biological agent.

Description

A method to efficiently knock out TIGIT gene in NK cells

technical field

The invention relates to a method for efficiently knocking out TIGIT gene in NK cells, and belongs to the technical field of gene editing.

Background technique

Natural killer cells (NK cells) are important immune cells in the body. They are derived from bone marrow lymphoid stem cells. Their differentiation and development depend on the bone marrow and thymus microenvironment. They are mainly distributed in bone marrow, peripheral blood, liver, spleen and lung. and lymph nodes. Different from T and B cells, NK cells are a type of lymphocytes that can non-specifically kill tumor cells and virus-infected cells without prior sensitization. It is not only related to anti-tumor, anti-viral infection and immune regulation, but also in some cases. It is involved in the occurrence of hypersensitivity reactions and autoimmune diseases, and can recognize target cells and killing mediators.

Studies have shown that although NK cells have powerful natural anti-tumor functions, and can recognize and attack tumor cells without pre-sensitization to tumor cell antigens (targets), the small number and low activity of activated NK cells in tumor tissues are the main reason for targeting solid tumors. The main obstacle to the efficacy of NK cell immunotherapy for tumors is that although high numbers and high purity NK cells can be obtained by in vitro expansion methods, only a small fraction of adoptively infused NK cells enter the solid tumor microenvironment through capillaries, contact and Killing tumor cells does not solve the problem of the low number of activated NK cells in tumor tissue; in addition, researchers have successively found that TIGIT (T cell immunoglobulin and ITIM domain) is one of the new immune checkpoints, which is associated with the ligand PVR/CD155 (Poliovirus receptor) binding can significantly inhibit the immune response of T cells or NK cells; the combined use of anti-TIGIT antibody and anti-PD-1 antibody can reverse the exhausted state of tumor-infiltrating CD8+ T cells and restore their anti-tumor immune activity .

In terms of NK cells, studies have also shown that exhausted NK cells express a large amount of TIGIT instead of PD-1. Whether knocking out the TIGIT gene in mice or blocking the TIGIT/PVR signaling pathway with antibodies can enhance the resistance of NK cells. Tumor activity and prolong survival of tumor-bearing mice. Although anti-TIGIT antibodies have shown promising efficacy in animal-level studies, clinical trials of TIGIT antibodies are few and far between. In a recent randomized, double-blind clinical trial, anti-TIGIT antibody combined with atezolizumab in first-line treatment of patients with PD-L1 positive non-small cell lung cancer, the objective response rate (ORR) of TIGIT antibody combined with atezolizumab ) was only 37%, which was only 16% higher than that of the placebo + atezolizumab group. It can be seen that the clinical efficacy of anti-TIGIT antibodies is still very limited, which may be related to the difficulty of the antibody to reach the local tumor, or to reach the local tumor. Antibodies are not sufficient to completely block the TIGIT/PVR signaling pathway, and this limitation largely restricts the development of TIGIT immune checkpoint blockade therapy.

Gene editing technology is an important technical means to study functional genomes. Four generations of gene editing technologies have been developed: meganucleases, zinc finger nucleases, transcription activator-like effector nucleases, CRISPR, etc. Efficient, cheap and widely used. CRISPR/Cas9 is a new type of nuclease system derived from prokaryotes. It is composed of two elements: guide sequence sgRNA and nuclease Cas9. At present, the CRISPR/Cas9 system has been successfully used in humans, mice, zebrafish, and Arabidopsis thaliana. , rice, Drosophila, silkworm and other species have achieved gene knockout, but so far there is no related report on the knockout of TIGIT gene in NK cells.

SUMMARY OF THE INVENTION

In view of the above problems existing in the prior art, the purpose of the present invention is to provide a method for efficiently knocking out TIGIT gene in NK cells, thereby obtaining NK cells that do not express TIGIT, so as to improve the recognition and killing activity of NK cells to tumor cells, It can be developed into safe and effective anti-tumor biological agents, and provide new biological agents for adoptive immunotherapy of tumors and viral infectious diseases (such as HIV/AIDS, novel coronavirus pneumonia/COVID-19).

For realizing the above-mentioned purpose of the invention, the present invention adopts following technical scheme:

A method for efficiently knocking out TIGIT gene in NK cells, the method is to use the CRISPR/Cas9 gene editing system to introduce the complex of sgRNA and Cas9 protein into the amplified NK cells by electroporation transfection, wherein: The sequence of the sgRNA is selected from any one of the sequences shown in SEQ ID NO.1-4.

In a preferred embodiment, the sequence of the sgRNA is selected from the sequence shown in SEQ ID NO.1 or 2.

In a preferred solution, in the complex, the mass ratio of sgRNA to Cas9 protein is 1:(3-5).

In a further preferred solution, in the complex, the mass ratio of sgRNA to Cas9 protein is 1:4.

In a preferred solution, the sgRNA is modified by phosphorylation.

In a further preferred solution, the sites for phosphorylation modification are: 3 thio groups and methoxy groups at the 3' end and the 5' end respectively.

In a preferred solution, the Cas9 protein is firstly codon-optimized in E. coli, and then constructed into the backbone of the Pet28a expression plasmid.

In a further preferred solution, the Cas9 protein has the sequence shown in SEQ ID NO.5.

One embodiment, the preparation of described sgRNA comprises the steps:

a) annealing, adding the upstream sequence of the TAGG endonuclease sticky end and the downstream sequence of the AAAC endonuclease sticky end to each sgRNA and performing annealing treatment together;

b) Linearization, the pUC57-sgRNA expression vector is linearized by BsaI;

c) connect, connect the annealed product to the pUC57-sgRNA expression vector linearized by BsaI, after the connected vector is transformed, picked and identified, the positive clone is shaken to extract the plasmid and measure the concentration;

d) PCR amplification, the sequence of the forward primer used is: TCTCGCGCGTTTCGGTGATGACGG, and the sequence of the reverse primer used is: AAAAAAAGCACCGACTCGGTGCCACTTTTTC;

e) In vitro transcription, the DNA of sgRNA was transcribed into RNA in vitro by using T7 transcription kit, and the RNA was recovered after identification by gel electrophoresis.

Compared with the prior art, the present invention has the following significant beneficial effects:

The research results of the present invention show that the method of the present invention can realize the knockout efficiency of TIGIT gene in NK cells close to 80%, not only the knockout efficiency is high, but also the obtained NK cells that do not express TIGIT can relieve CD155+ tumor cells The immunosuppressive effect on NK cells, compared with wild-type NK cells, showed stronger anti-tumor activity in both in vivo and in vitro experiments, significantly improved the recognition and killing activities of NK cells on tumor cells, and is expected to be developed into a safe Effective anti-tumor biological agents have obvious application prospects and clinical application value, and are of great significance for adoptive immunotherapy of tumors and viral infectious diseases (such as HIV/AIDS, novel coronavirus pneumonia/COVID-19).

Description of drawings

Figure 1 shows the comparison of viable cell percentages of NK cells after electroporation of CM137 and EN138, and the control group is NK cells without electroporation;

Figure 2A shows the comparison of the percentage of viable cells of NK cells after electroporation of different sgRNA/Cas9 protein complexes using the CM137 program.

Figure 2B shows the comparison of the knockout efficiency of TIGIT gene after electrotransduction of different sgRNAs targeting TIGIT, in which CD56 is a surface marker of human NK cells, and the WT group is NK cells only electrotransduced with Cas9 protein but not electrotransduced with sgRNA;

Figure 3A shows the comparison of the cell activity of NK cells after electroporation of unmodified and phosphorylated sgRNA-2 sequences targeting TIGIT using the CM137 program. NK cells;

Figure 3B shows the comparison of the knockdown efficiency of TIGIT gene after electroporation of unmodified and phosphorylated sgRNA-2 sequences targeting TIGIT, in which CD56 is a surface marker of human NK cells, and the WT group is only NK cells electroporated with Cas9 protein but not electroporated with sgRNA;

Figure 3C is a comparison of the sequencing analysis results after electroporation of unmodified and phosphorylated sgRNA-2 sequences targeting TIGIT;

Figure 4 shows the effect of NK cells after TIGIT knockout and wild-type NK cells on H1975 lung cancer, HCT8 colorectal cancer, PC3 prostate cancer, LM7 osteosarcoma, HepG2 liver cancer, A375 and melanoma, MDA-MB-231 breast cancer, Comparison of in vitro killing activity of K562 leukemia cells;

Figure 5 shows the comparison of the degranulation level of NK cells after knockout of TIGIT gene and wild-type NK cells compared with H1975 lung cancer cells or HepG2 liver cancer cells in vitro;

Figure 6A shows the comparison of the control of tumor growth in lung cancer tumor-bearing mice by adoptive immunotherapy of NK cells after knockout of TIGIT gene and wild-type NK cells;

Figure 6B shows the effect of adoptive immunotherapy of different types of NK cells on the body weight of tumor-bearing mice.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In the following examples, the experimental methods without specific conditions are usually in accordance with conventional conditions or in accordance with the conditions suggested by the manufacturer.

Example 1

A method for efficiently knocking out TIGIT gene in NK cells, comprising the following specific steps:

1. Expansion and culture of NK cells

1) Take out the cryopreserved human peripheral blood mononuclear cells (PBMC) from liquid nitrogen and thaw rapidly in a 37°C water bath;

2) Add 4 mL of RPMI-1640 complete culture solution containing 10% FBS and 1% penicillin/streptomycin double antibody to a new 15 mL centrifuge tube, and transfer 1 mL of PBMC suspension to a 15 mL centrifuge tube;

3) Centrifuge at 250 × g for 5 min at room temperature;

4) Discard the supernatant and resuspend the cells with 1 mL of RPMI-1640 medium;

5) Add 19mL RPMI-1640 culture medium to a new 75mL cell culture flask, and transfer the above cell suspension to the culture flask;

6) Add human recombinant IL-2 protein to the culture flask, and the final concentration is 200U/mL;

7) Place the culture flask in a 37°C, 5% CO ₂ incubator, and after culturing overnight, count the PBMCs in the culture flask;

8) Take out the cryopreserved irradiated EK562 engineered cells from liquid nitrogen, and rapidly thaw them in a 37°C water bath;

9) Add 4mL of RPMI-1640 complete culture medium to a new 15mL centrifuge tube, and transfer the EK562 cell suspension to a 15mL centrifuge tube;

10) Centrifuge at 250 × g for 5 min at room temperature;

11) Discard the supernatant, use 1 mL of RPMI-1640 medium to resuspend the cells and count the cells;

12) Add EK562 cells to the culture flask according to the effect-target ratio of PBMC:EK562=1:1;

13) Culture the cells in a 37°C, 5% CO ₂ incubator, change the medium every two days and count the cells, and control the cell density within the range of 0.5 to 1×10 ⁶ cells/mL, depending on the volume of the culture medium Add IL-2, the final concentration is 100U/mL;

14) When culturing to the 7th day, count the cells in the culture flask, and add EK562 cells to the culture flask again according to the effect-target ratio of PBMC:EK562=1:1;

15) Culture the cells in a 37°C, 5% CO ₂ incubator, change the medium every two days and count the cells, and control the cell density within the range of 0.5 to 1×10 ⁶ cells/mL, depending on the volume of the culture medium Add IL-2, the final concentration is 100U/mL;

16) After culturing to the 14th day, the purity of NK cells in the culture flask can reach more than 95%.

2. Preparation of sgRNA

1) Annealing, to each sgRNA (the sequence of the sgRNA is selected from any one of the sequences shown in SEQ ID NO. 1 to 4) adding the upstream sequence of the TAGG endonuclease sticky end and the downstream adding the AAAC endonuclease sticky end After the sequence, annealed together through the program (95°C, 5min; 95°C-85°C, -2°C/s; 85°C-25°C, -0.1°C/s; hold at 4°C);

2) Linearization, the pUC57-sgRNA expression vector (Addgene#51132) was linearized by BsaI (NEB: R0539L). The linearization system was: pUC57-sgRNA expression vector 2 μg; buffer solution (NEB: R0539L) 5 μL; BsaI 1 μL; ddH2O Make up to 50μL;

3) Ligation, ligating the annealed product to the pUC57-sgRNA expression vector linearized by BsaI, the ligation system is: 1 μL of T4 ligation buffer solution (NEB: M0202L), 20 ng of linearized vector, 5 μL of annealed oligo fragment (10 μM), T4 ligase (NEB: M0202L) 0.5 μL, filled with ddH ₂ O to 10 μL, and ligated at 16°C for one hour; the ligated vector was transformed, picked, and identified, and the positive clones were shaken to extract plasmids (Axygene: AP-MN) -P-250G) and determine the concentration;

4) PCR amplification, the sequence of the forward primer sgRNA-F used is: TCTCGCGCGTTTCGGTGATGACGG, and the sequence of the reverse primer sgRNA-R used is: AAAAAAAGCACCGACTCGGTGCCACTTTTTC; the sgRNA sequence containing the T7 promoter is amplified by PCR and identified by gel electrophoresis After the band was treated with RNA-SECURE (Thermo, #AM7005) at 65°C for 15 minutes, then the amplification product was recovered (Axygene: AP-PCR-250G) and the concentration was determined;

5) In vitro transcription, using T7 transcription kit (Thermo, #AM1354) to transcribe the DNA of sgRNA into RNA in vitro, after identification by gel electrophoresis, use RNA recovery (Thermo, #AM1908) to recover the sgRNA to measure the concentration.

3. Preparation and purification of Cas9

1) Codon optimization, the Cas9 protein is subjected to codon optimization of E. coli, and then the sequence is synthesized and constructed into the Pet28a expression plasmid backbone, and the optimized sequence is shown in SEQ ID NO.5;

2) Plasmid transformation, the Pet28a-Cas9 plasmid was transformed into a plate with BL21 competence, and a single clone was picked for expression by shaking the bacteria. Prepare 2 L of culture medium at 37°C and shake the bacteria at 220 rpm, and wait for the OD value of the bacterial solution to be 0.6-0.8 IPTG 2mL (2M concentration) was added between, and the bacteria were harvested after 24 hours of induction culture;

3) Collect bacteria, centrifuge the bacteria liquid at high speed: 4000rpm for 30min, discard the supernatant;

4) Bacteria break, after the centrifuged bacteria are blown away with Buffer A, protease inhibitors are added, Escherichia coli is broken with a high-pressure crusher, and the supernatant is taken after high-speed centrifugation; the formula of the Buffer A is: 25mM Tris PH =8.0, 500 mM NaCl, 10% (v/v) glycerol, 0.22 μM filter;

5) Pass the column, filter the crushed supernatant with a 0.45 μM filter membrane, and add a cobalt ion affinity chromatography column (Clontech, 635504) after rinsing with Buffer A to adsorb the His-tagged Cas9 protein;

6) To remove impurities, use 40 mL of Buffer A supplemented with 5 mM imidazole to pass through the column to remove impurities with lower affinity;

7) Elution, use 30 mL of buffer A added with 500 mM imidazole to pass through the column to replace the target protein Cas9;

8) Concentrate, add the eluted target protein to the protein concentration column after Western blot identification, 3900rpm, 20min;

9) The concentrated protein is subjected to ion exchange chromatography (IEC) to remove the nucleic acid bound to the protein, and then concentrated again.

4. Transfection of NK cells by electroporation

1) Mix Cas9 protein and sgRNA at a mass ratio of 4:1, gently pipette and mix with a pipette tip, and place at room temperature for 15 minutes;

2) Prepare cells, wash NK cells with PBS and centrifuge;

3) Electroporation, prepare electroporation solution with electroporation kit (Lonza, V4XP-3032 or V4XP-3024), use electroporation solution to resuspend cells and RNP (ie: the complex of sgRNA and Cas9 protein), add In the electroporation cup, be careful not to have air bubbles. Select the electroporation program to perform electroporation. After electroporation, use a pipette tip to aspirate the liquid and put it into the pre-warmed medium for culture. Finally, culture as needed for testing. Figure 1 shows the comparison of the percentage of viable NK cells after electroporation between CM137 and EN138. It can be seen from the results shown in Figure 1 that the CM137 electroporation program has less effect on cell viability.

Example 2

Gene knockout efficiency was detected by flow cytometry:

1) Collect wild-type or TIGIT knockout NK cells and wash twice with 1×PBS containing 1% fetal bovine serum (FBS);

2) Resuspend the cells in PBS containing 1% FBS and count, and adjust the cell concentration to 3×10 ⁶ cells/mL;

3) Add 50 μL of the above cell suspension into a new 1.5 mL centrifuge tube, add 1 μL of PE-anti TIGTI antibody, and incubate at 4°C for 30 min in the dark;

4) Wash twice with 1% FBS 1×PBS;

5) Resuspend the cells with 300 μL of 1% FBS 1×PBS and perform flow cytometry detection.

The detection results are shown in Figures 2A and 2B. From the results shown in Figures 2A and 2B, it can be seen that: sgRNA-1 and 2 have higher knockout efficiency than sgRNA-3 and 4, and sgRNA-2 has higher knockdown efficiency than sgRNA-1. Vitality has a smaller effect, as shown in Table 1:

Table 1 Detection of TIGIT knockout efficiency by flow cytometry

name Percentage of CD56+/TIGIT+ NK cells (%) Knockout rate (%) Percentage of viable cells (%) WT 75.3 0 52.4 TIGIT-1 58.2 17.1 29.7 TIGIT-2 58.7 16.6 46.0 TIGIT-3 75.6 0 40.5 TIGIT-4 72.8 2.5 54.7

From the results in Table 1, it can be seen that under the same conditions, the sgRNA selected from the sequence shown in SEQ ID NO. 1 or 2 has a higher knockout efficiency of the TIGIT gene in NK cells, and has less effect on cell viability.

Example 3

The phosphorylation-modified gRNA targeting TIGIT was purchased from GenScript Biotechnology Co., Ltd., and the specific phosphorylation sites were: 3 thio groups and methoxy groups at the 3' and 5' ends respectively.

The specific sequence is TIGIT-2: CTGGTGTCTCCTCCTGATCT

Figure 3A shows the comparison of the cell activity of NK cells after electroporation of unmodified and phosphorylated sgRNA-2 sequences targeting TIGIT using the CM137 program. NK cells;

Figure 3B shows the comparison of the knockdown efficiency of TIGIT gene after electroporation of unmodified and phosphorylated sgRNA-2 sequences targeting TIGIT, in which CD56 is a surface marker of human NK cells, and the WT group is only NK cells electroporated with Cas9 protein but not electroporated with sgRNA;

Figure 3C is a comparison of the sequencing analysis results after electroporation of unmodified and phosphorylated sgRNA-2 sequences targeting TIGIT;

From the results shown in Figure 3A to Figure 3C, it can be seen that the percentage of CD56+/TIGIT+NK cells in wild-type NK cells is 91.3%, and the phosphorylation-modified TIGIT-2 gRNA can reduce the percentage of CD56+/TIGIT+NK cells to 12.9%, while The unphosphorylated TIGIT-2 gRNA could only be reduced to 68.8%, indicating that the phosphorylated sgRNA has better stability and stronger activity.

As shown in Figure 3C, the genome was extracted from the cells and subjected to sanger sequencing. The results were consistent with the flow cytometry results, which further verified that the phosphorylation-modified sgRNA had a higher knockout efficiency. The specific results are shown in Table 2:

Table 2 Detection of TIGIT-2 knockout efficiency by flow cytometry

Example 4

1. Bioluminescence assay to detect the killing activity of NK cells in vitro

1) Collect tumor cells and NK cells for counting, add tumor cells to a 96-well plate, 10,000 cells/well, and the final volume is 50 μL;

2) Collect wild-type and TIGIT gene knockout NK cells, count them according to the ratio of NK cells:tumor cells (abbreviated as E:T) = 0.5:1, 1:1 and 2:1, and transfer them to a 96-well plate. Add 50 μL of NK cells to the

3) Make three replicate wells for each group, and set up control wells for tumor cell culture alone and NK cell culture alone;

生物发光检测试剂(购自Promega公司)；4) After culturing for 24 hours, add 100 μL to each well

Bioluminescence detection reagent (purchased from Promega);

5) After shaking at room temperature for 1min, let stand for 10min, and then detect the bioluminescence intensity with a microplate reader;

6) Calculate the killing activity of NK cells according to the following formula:

Mean _Mix : the average luminescence intensity of the co-culture group of NK cells and tumor cells;

Mean _NK : the average luminescence intensity of NK cells cultured alone;

Mean _Tumor : Mean luminescence intensity of tumor cells cultured alone.

As shown in Figure 4, after 24 hours of co-culture of wild-type or TIGIT gene knockout NK cells and tumor cells, the percentage of NK cells killing target cells was detected by bioluminescence method, and the results showed that the NK cells after TIGIT gene knockout Compared with wild-type NK cells, they have stronger anti-tumor activity, and the specific results are shown in Table 3:

Table 3 Comparison of antitumor activity of NK cells after TIGIT gene knockout and wild-type NK cells

2. Detection of NK cell degranulation level

1) After the tumor cells were digested, the cells were counted, and the cell concentration was adjusted to 3×10 ⁶ cells/mL;

2) Take wild-type or TIGIT gene knockout NK cells, wash twice with RPMI-1640 complete medium and count, and adjust the cell concentration to 3×10 ⁶ cells/mL;

3) Take 100 μL of the above NK cells and add them to a new 48-well plate, and then add 100 μL of RPMI-1640 complete culture medium to form a single NK cell group;

4) Take 100 μL of each of the above tumor cells and NK cells and add them to a 48-well plate, which is the NK+ tumor cell group;

5) Add 5 μL PE/Cy5-anti CD107A antibody or the corresponding isotype control antibody to the above wells, and place in a 37°C, 5% CO ₂ incubator for incubation;

6) After culturing for 4 hours, collect cells from each well, transfer them to a new 1.5 mL centrifuge tube, and wash once with 1% FBS 1×PBS;

7) Resuspend cells with 50 μL 1% FBS 1×PBS, add 1 μL FITC-anti CD56 antibody or corresponding isotype control antibody to each tube, and incubate at 4°C for 30 min;

8) Wash twice with 1% FBS 1×PBS, add 300 μL of 1% FBS 1×PBS to resuspend the cells and perform flow cytometry detection.

Wild-type or TIGIT knockout NK cells were co-cultured with tumor cells, and the fluorescence intensity of CD107A in NK cells was detected by flow cytometry, which could indirectly reflect the degranulation level of NK cells. The higher the percentage of CD107A-positive NK cells, the more activated NK cells. Degranulation is one of the important means for NK cells to induce tumor cell death. It can be seen from Figure 5 that the positive percentage of CD107A of NK cells cultured alone does not exceed 3%, indicating that they are in a resting state, and the positive rate of CD107A will be greatly increased only when NK cells are co-cultured with tumor cells. Compared with wild-type NK cells, the positive rate of CD107A in NK cells after TIGIT gene knockout was significantly increased, indicating that NK cells after TIGIT gene knockout were more active in recognizing and killing tumor cells.

3. In vivo detection of antitumor activity of NK cells after knockout of TIGIT gene

1) Severe combined immunodeficiency (SCID-bg) mice with deletion of T, B and NK cells were selected as tumor-bearing mice. The ordered mice were all male, 5 weeks old, and the mice were raised in SPF animal breeding rooms. After one week of adaptive feeding, tumor cells were inoculated into its back;

2) Digest and collect H1975 lung cancer cells, and use serum-free RPMI-1640 medium to resuspend the cells;

3) Adjust the concentration of H1975 cell suspension to 3×10 ⁷ cells/mL;

4) 6-week-old male SCID-Bg mice were weighed and anesthetized by intraperitoneal injection of sodium pentobarbital at a dose of 25 mg/kg body weight;

5) Use a shaver to shave off the hair on the right side of the mouse's back near the scapula, and inject 100 μL of the above cell suspension subcutaneously;

6) After 3 days of inoculation, mice were randomly divided into 2 groups according to body weight, with 5 mice in each group, respectively receiving wild-type NK cells and TIGIT knockout NK cells adoptive immunotherapy: NK cells were administered by tail vein injection, and each mouse was injected 1×10 ⁷ NK cells were injected once a week; the tumor size of mice was measured every 3 days, and the body weight of mice was recorded at the same time.

As shown in Figure 6A, after treatment with NK cells after TIGIT gene knockout (TIGIT-KO), the tumor growth of lung cancer tumor-bearing mice was significantly inhibited. Compared with the WT group, the volume of tumor mass in mice was reduced by 57%, as shown in Table 4:

Table 4 Tumor volume of tumor-bearing mice

It can be seen from the results in Figure 6A and Table 4 that the NK cells after knocking out the TIGIT gene have stronger anti-tumor effects in vivo. In addition, it can be seen from Figure 6B that after adoptive immunotherapy of NK cells in tumor-bearing mice, there was no significant difference in the body weight of the two groups of mice, and the body weight of the mice increased slowly over time, indicating that the NK cells after the knockout of the TIGIT gene are similar to As safe and effective as wild-type NK cells.

To sum up, it can be seen that the NK cells obtained by the present invention after knocking out the TIGIT gene have stronger anti-tumor activity than ordinary NK cells in vivo and in vitro, and the NK cells after knocking out the TIGIT gene have good anti-tumor activity in vivo. Therefore, the NK cells obtained by the present invention after knocking out the TIGIT gene are expected to be developed into safe and effective anti-tumor biological preparations, and have obvious application prospects and clinical application value.

Finally, it is necessary to explain here: the above embodiments are only used to further describe the technical solutions of the present invention in detail, and should not be construed as limiting the protection scope of the present invention. Non-essential improvements and adjustments belong to the protection scope of the present invention.

Claims (5)

1. A method for efficiently knocking out TIGIT gene in NK cells for non-therapeutic purposes, the method is to use CRISPR/Cas9 gene editing system to introduce the complex of sgRNA and Cas9 protein into amplified NK by electroporation transfection In the cell, it is characterized in that: the sequence of the sgRNA is selected from the sequence shown in SEQ ID NO.1 or 2, and the sgRNA undergoes phosphorylation modification, and the sites for phosphorylation modification are 3 ' end and 5 ' end respectively. thio and methoxy. 2 . The method according to claim 1 , wherein in the complex, the mass ratio of sgRNA to Cas9 protein is 1:(3-5). 3 . 3 . The method according to claim 1 , wherein the Cas9 protein is first optimized by E. coli codons, and then constructed into the backbone of the Pet28a expression plasmid. 4 . 4. The method according to claim 3, wherein the Cas9 protein has the sequence shown in SEQ ID NO.5. 5. method according to claim 1, is characterized in that, the preparation of described sgRNA comprises the steps: a) annealing, adding the upstream sequence of the TAGG endonuclease sticky end and the downstream sequence of the AAAC endonuclease sticky end to each sgRNA and performing annealing treatment together; b) Linearization, the pUC57-sgRNA expression vector is linearized by BsaI; c) connect, connect the annealed product to the pUC57-sgRNA expression vector linearized by BsaI, after the connected vector is transformed, picked and identified, the positive clone is shaken to extract the plasmid and measure the concentration; d) PCR amplification, the sequence of the forward primer used is: TCTCGCGCGTTTCGGTGATGACGG, and the sequence of the reverse primer used is: AAAAAAAGCACCGACTCGGTGCCACTTTTTC; e) In vitro transcription, the DNA of sgRNA was transcribed into RNA in vitro by using T7 transcription kit, and the RNA was recovered after identification by gel electrophoresis.

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