CN111321169A

CN111321169A - Genetically modified NK cell and preparation method and application thereof

Info

Publication number: CN111321169A
Application number: CN202010158055.0A
Authority: CN
Inventors: 黄常新; 高岚岚; 杨丽丽; 施燕飞; 王聪洁; 张嗣玉; 葛转敏; 苏萌; 李永强
Original assignee: Hangzhou Normal University
Current assignee: Hangzhou Normal University
Priority date: 2020-03-09
Filing date: 2020-03-09
Publication date: 2020-06-23

Abstract

The invention discloses genetically modified NK cells and a preparation method and application thereof. The preparation method of genetically modified NK cells comprises: (a) linking the chemokine receptor gene CXCR2 gene and IL2 gene to a lentiviral vector to form a recombinant genetic engineering vector; (b) transfecting the recombinant genetic engineering vector into the NK92 cells to obtain genetically modified NK cells. The genetically modified NK cells highly express CXCR2 and IL2, which can well inhibit tumor growth.

Description

Genetically modified NK cell and preparation method and application thereof

Technical Field

Embodiments of the present disclosure relate to the field of biomedicine, and more particularly, to genetically modified NK cells and methods of making and using the same.

Background

At present, adoptive cell immunotherapy of killing tumor cells by culturing in vitro and reinfusing autologous or allogeneic immunocompetent cells in vivo has become an important tumor therapy approach. Solid tumor cells have high heterogeneity, complex tissue components and lack of ideal specific targets. Natural Killer (NK) cells have no Major Histocompatibility Complex (MHC) restriction, and can recognize and attack tumor cells without pre-sensitization of tumor cell antigens (targets), thereby avoiding the obstacle of lack of ideal targets of solid tumor cells. And the activated NK cells can rapidly kill the diseased cells through direct cytotoxic action on one hand, and can indirectly kill the diseased cells by secreting cytokines to regulate body-related immune cells on the other hand, so that the NK cells show huge application potential in tumor immunotherapy.

Compared with the NK cells of autologous sources, the NK cells of healthy allogeneic sources show better anti-tumor effect, become the mainstream of the recent clinical research, and have been applied to the treatment of various tumors including leukemia, lymphoma, osteosarcoma and the like. Miller et al showed that the reinfusion of allogeneic NK cells was well tolerated and safe, without causing serious side effects and graft versus host reaction (GVHD). However, its therapeutic effect in other solid tumors including colon cancer is not ideal. Only a small part of NK cells can enter a solid tumor microenvironment through capillaries to contact and kill tumor cells, the number of effector cells which can be stimulated and converted into the NK cells in peripheral blood of a tumor patient is small, the number of the NK cells is reduced, and meanwhile, the expanded autologous NK cells are low in function due to different degrees of damage to the function.

Meanwhile, tumor cells have evolved multiple mechanisms to escape NK cell recognition, including expression of mhc class i molecules and NK inhibitory receptor ligands. Chimeric receptors have been extensively studied in order to overcome the resistance of tumor cells to NK cell-mediated solubility and to improve tumor recognition. The NK cells are genetically modified to express a chimeric receptor specific for a Tumor Associated Antigen (TAA), which signal activates the NK cells upon exposure to the antigen. The introduction of the chimeric antigen receptor will largely avoid the transmission of inhibitory signals into NK cells, thereby enhancing NK cell anti-tumor capacity. NK cells are considered to be a competitive CAR vector. Firstly, the survival period of NK cells in vivo is short, and unlike T cells, the NK cells do not cause long-term side effects due to the continuous existence in vivo; second, NK cell activation is associated with tumor cell surface associated ligands in addition to Chimeric Antigen Receptor (CAR) mediation, avoiding or reducing the risk of damage to normal cells including off-target effects. Although CAR-NK cells can continuously proliferate, tumor chemotaxis function is not enhanced, and the actual effect of CAR-NK is not ideal because solid tumors lack ideal targets.

The small number and low activity of activated NK cells in tumor tissues are the main obstacles of the current NK cell immunotherapy aiming at solid tumors. It has been shown that NK cells are directed into tumors mainly through the interaction of chemokine receptors on their surface with chemokines produced by tumor secretion. Preclinical studies have shown that various cytokines including IL2, IL15, IL18, IL21, etc., have the effects of promoting NK cell proliferation and enhancing NK cell function. At present, most of the existing technical schemes are to give exogenous cytokines or improve the expression level of chemokine receptors through a transgenic technology, so that the proliferation and the activity of NK cells are promoted, and the number of NK cells in tumor bodies is increased. The disadvantages of these approaches are that systemic application of exogenous cytokines has significant toxic side effects on the body and the actual concentration on NK cells is not high. The maintenance time of overexpression by using a transgenic technology is short, the expression amount of the cell factor is difficult to control, and more importantly, the application of the transgenic modification mode is limited due to the limited control of Major Histocompatibility Complex (MHC) molecules.

Disclosure of Invention

In order to solve the problems, the NK cell is modified by applying a CRISPR-CAS9 gene editing technology, the chemotactic effect of the NK cell to a tumor body is enhanced, the sustainable proliferation of the NK is realized, the tumor killing activity of the NK cell is improved, the problem of insufficient NK cell quantity in a tumor body tissue is solved, and a local immune microenvironment is activated.

The invention provides a preparation method of genetically modified NK cells, which comprises the following steps: NK cells are enabled to highly express CXCR2 and IL2 by means of genetic engineering.

In the preparation method, the genetic engineering means is realized by a transcription activated CRISPR-CAS9 system.

In the above preparation method, in the CRISPR-CAS9 system, sgRNA target sequences for CXCR2 are: GCGAGTTCAGACAAGTCCGT, as shown in SEQ ID NO. 1; the sgRNA target sequence for IL2 was: GTGGGCTAATGTAACAAAGA, as shown in SEQ ID NO. 3.

In the above preparation method, the genetic engineering operation comprises the steps of: (1) construction of sgRNA vector: constructing a lentiviral vector comprising the sgRNA target sequence for CXCR2 and the sgRNA target sequence for IL 2; (2) construction of dCas9 plasmid: synthesizing a dCas9 plasmid, wherein the dCas9 plasmid has a nuclear localization signal, the C-terminal of the dCas9 plasmid has a VP64 transcription activation domain, the dCas9 plasmid has double mutations of D10A and H840A, and the dCas9 plasmid has no enzyme cutting activity; (3) infection of NK cells: transfecting 293T cells with the sgRNA vector and the dCas9 plasmid to prepare lentivirus solution, and infecting NK cells by combining virus stock after concentrating PEG8000 to obtain the genetically modified NK cells.

In the above preparation method, the transfection of 293T cells was performed by liposome-mediated transfection, and the infection of NK cells was performed by lentivirus infection.

The invention also provides a genetically modified NK cell, wherein the genetically modified NK cell is prepared by the preparation method.

The present invention also provides a genetically modified NK cell that overexpresses CXCR2 and IL 2.

The invention also provides an sgRNA applied to a transcription activated CRISPR-CAS9 system, wherein a target sequence of the sgRNA comprises: sgRNA target sequence for CXCR2 and sgRNA target sequence for IL2, sgRNA target sequence for CXCR2 is: GCGAGTTCAGACAAGTCCGT, as shown in SEQ ID NO. 1; the sgRNA target sequence for IL2 was: GTGGGCTAATGTAACAAAGA, as shown in SEQ ID NO. 3.

The invention also provides a kit, and the kit contains the sgRNA.

The present invention also provides the use of the genetically modified NK cell as described above for the prevention, inhibition or treatment of a tumor.

The method modifies the NK cells through a gene editing technology, so that the NK cells stably over-express chemokine receptors CXCR2 and interleukin-2, the number of the NK cells chemotactic to a tumor body is obviously increased by strengthening the chemotactic process of the NK cells, and the killing activity of the NK cells is greatly improved; by strengthening the autocrine action of interleukin-2 of NK cells, the NK cells chemotactic to tumor bodies can be continuously proliferated, and the killing activity of the NK cells is further improved.

Drawings

The above and other features, advantages and aspects of various embodiments of the present disclosure will become more apparent by referring to the following detailed description when taken in conjunction with the accompanying drawings. Throughout the drawings, the same or similar reference numbers refer to the same or similar elements. It should be understood that the drawings are schematic and that elements and features are not necessarily drawn to scale.

Fig. 1 shows a schematic diagram of a method of implementing the present disclosure.

FIG. 2 shows a plasmid map of pHS-ACR-LW 415.

FIG. 3 shows a plasmid map of pHS-ACR-LW 649.

Fig. 4 shows a schematic diagram of the working principle of CRISPR-CAS 9.

Detailed Description

Embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While certain embodiments of the present disclosure are shown in the drawings, it is to be understood that the present disclosure may be embodied in various forms and should not be construed as limited to the embodiments set forth herein, but rather are provided for a more thorough and complete understanding of the present disclosure. It should be understood that the drawings and embodiments of the disclosure are for illustration purposes only and are not intended to limit the scope of the disclosure. Unless otherwise indicated, all reagents used in the present disclosure are conventional.

The current application of exogenous cytokines (such as IL2) has great side effects on the body, including fever, nausea, vomiting, renal insufficiency, edema, hypotension and the like. In addition, exogenous cytokines do not act directly on NK cells at high concentrations. Furthermore, the chemotactic ability of NK cells towards tumor tissue was not enhanced.

The invention aims to provide a novel efficient tumor chemotactic sustainable-proliferation anti-tumor NK cell and a preparation method thereof. Through over-expressing the chemotactic factor receptor and the cytokine on the surface of the NK cell, the chemotactic process of the NK cell to a tumor body is strengthened, the continuous proliferation of the NK cell is realized, and the tumor killing activity of the NK cell is improved, so that the aims of increasing the number of the NK cells at the local part of the tumor body and enhancing the activity are fulfilled, and the main problems of restricting the curative effect of NK cell immunotherapy at present are solved: 1. the number of NK cells infiltrating into tumor body tissues in the solid tumor is insufficient; 2. the NK cell activity of the tumor patients is low.

The low number and activity of activated NK cells in tumor tissues are the main obstacles for the curative effect of NK cell immunotherapy. The invention uses CRISPR-CAS9 gene editing technology to ensure that NK cells are constantly over-expressed (can be inherited to descendant NK cells) chemokine receptors CXCR2 and interleukin-2 (IL2), enhances the chemotactic effect of the NK cells to tumor tissues, and continuously acts on the NK cells through an autocrine way (the NK cells act on the NK cell membrane receptors after being secreted out of the cells, namely a secretion system and an action system are in the same NK cells), so that the NK cells are continuously amplified and activated in the tumor tissues, the problem of insufficient NK cell number in the tumor body tissues is solved, the killing activity of the NK cells is improved, and local immune microenvironment is activated.

Fig. 1 shows a technical route diagram of the present invention. The following describes the brief steps of the method of the invention: constructing an SSA (Single-strand annealing) report vector, connecting the SSA report vector with an sgRNA-Cas9 plasmid by using T4 DNA ligase, constructing a lentiviral vector thereof after high-purity extraction to transfect NK92 cells, and using a plasmid for expressing 'D10A and H840A double-mutant enzyme-free active Cas9 protein with nuclear localization signals and a VP64 transcription activation structural domain at the C-terminal thereof'; sequences of 5KB DNA fragments upstream of transcription start sites of CXCR2 and IL2 genes are determined by a database of NCBI, and sgRNAs are designed and synthesized. CXCR2-IL2 gRNA sequence, the middle is expressed by 2A gene interval, the frame selects PUC57 (containing puro (puromycin) resistance, GFP green fluorescent protein and other elements), the double chains are combined with Cas9 expression plasmid linearized after being cut by BbsI endonuclease, the sequencing verification is carried out, expression gRNA plasmid (LW649) is constructed through T4 ligase, the expressed sgRNA is bound with VP64-Cas9 expressed by plasmid LW415 after being expressed, and the plasmid map of Phs-ACR-LW415 is shown in FIG. 2. The sgRNA can guide Cas9 nuclease to recognize and cut a target DNA sequence, a structural domain with nuclease activity in a Cas9 protein is mutated to obtain a Cas9(dCas9) without nuclease activity, the dCas9 losing the nuclease activity after site-directed mutation fuses a transcription activation domain (VP64), the expressed fusion protein is guided by the sgRNA to target and recognize a target gene promoter region, and the fusion expressed transcription activation domain or transcription regulation domain recruits related transcription factors, so that the expression of a target gene is accurately and specifically regulated.

In some embodiments, the genetically engineered means is achieved by the transcription-activated CRISPR-CAS9 system. Fig. 4 shows a schematic diagram of the working principle of CRISPR-CAS 9.

In some embodiments, in the CRISPR-CAS9 system, the sgRNA target sequence for CXCR2 is: GCGAGTTCAGACAAGTCCGT, as shown in SEQ ID NO.1 of Table 1; the sgRNA target sequence for IL2 was: GTGGGCTAATGTAACAAAGA, as shown in SEQ ID NO.3 of Table 1.

In some embodiments, the step of genetically engineering is:

(1) construction of sgRNA vector: constructing a lentiviral vector comprising the sgRNA target sequence for CXCR2 and the sgRNA target sequence for IL 2;

(2) construction of dCas9 plasmid: synthesizing a dCas9 plasmid, wherein the dCas9 plasmid has a nuclear localization signal, the C-terminal of the dCas9 plasmid has a VP64 transcription activation domain, the dCas9 plasmid has double mutations of D10A and H840A, and the dCas9 plasmid has no enzyme cutting activity;

(3) infection of NK cells: transfecting 293T cells with the sgRNA vector and the dCas9 plasmid to prepare lentivirus solution, and infecting NK cells by combining virus stock after concentrating PEG8000 to obtain the genetically modified NK cells.

In some embodiments, the transfection of the 293T cells is by liposome-mediated transfection and infection of NK cells is by lentiviral infection.

The steps of the present invention are further illustrated below:

(1) a plasmid expressing "enzymatically inactive Cas9 double mutated at its C-terminus with VP64 transcription activation domain with nuclear localization signal, D10A and H840A, with plasmid name: LW415, purchased from Synbiotic Gene, Beijing; the goods number is: A-FW-201804200137;

(2) the sgRNA is designed and synthesized by obtaining a 5KB DNA fragment sequence upstream of the transcription start site of CXCR2 and IL2 target genes from a database of the national information technology center (NCBI), the activated gRNA sequence with better selective effect and specificity is scored according to http:// crispr-era.Stanford, the off-target effect is detected, a syngeneic gene company is entrusted to synthesize CXCR2-IL2 gRNA, cDNA is formed by PCR amplification through 2A gene (the sequence is SEQ ID NO. 5: GGATCTGGCGCCACCAACTTCTCTCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCAGGCCCA) interval, a slow virus vector (pHS-ACR-LW649 (the figure 3 shows the plasmid map of Phs-ACR-LW 649) is constructed by combining T4 DNA ligase with a linearized framework PUC57vetcor, pLV-hU6-CXCR2 sgRNA is (pLV-Kbac-7 SK-IL2 sgRNA-hef sgRNA-631-DH 23), and the expression state is transformed by pLV-4 DNA ligase, and (4) picking a single clone, carrying out small extraction on the plasmid, and carrying out sequencing verification. High purity extracted plasmid, SSA reporter vector (backbone PUC57 plasmid) purchased from synbiotic gene company, beijing; the goods number is: A-FW-201807200313.

(3) 293T cells are cultured, the 293T cells are transfected by Lipo3000, the transfection ratio is 6-well plates, the A solution comprises Lipo 30007.5 mu l, α -MEM (serum-free) 242.5 mu l, the B solution comprises α -MEM medium, ps PAX 23 mu g, PMD2G 1 mu g, LW 4154 mu g, LW 6494 mu g and P300016 mu l (2 mu l/mu g) are prepared into mixed solution, the B solution is added into the A solution after 5 minutes and mixed evenly, the 293T cell culture solution is added evenly after 20 minutes and mixed evenly, the fluorescence effect is observed under a fluorescence microscope after 48 hours and 96 hours, the slow virus solution is collected and centrifuged for 20 minutes at 4 ℃ and 3000g, the supernatant is taken, the PEG8000 concentrated virus solution is used, the titer is detected by an immunofluorescence method, the infection concentration is determined, the NK92 cells are infected, and the puro resistance is screened.

(4) qPCR and ELISA verify gRNA activity, and detect the expression levels of CXCR2 and IL2 target genes.

(5) The in vitro chemotactic capacity and killing and proliferation activity of the gene modified NK cells are detected by a Transwell method and a CCK8 method.

(6) The chemotaxis and proliferation activity of the gene modified NK cell in vivo is verified by establishing a human colon cancer tumor model through SCID mice.

The invention develops the tumor chemotactic sustainable-proliferation anti-tumor allogeneic NK cell, and well solves the defect of autologous NK cells. The CRISPR-CAS9 gene editing technology overcomes the defects of short overexpression time, difficult control of expression quantity, low cytokine level directly acting on NK cells and the like of a transgenic scheme.

NK cells have powerful and unique anti-tumor advantages, and the anti-tumor activity of the NK cells can identify and attack tumor cells without tumor cell antigens (targets). However, the therapeutic effect of NK cells on solid tumors including colon cancer is not ideal, and one of the limiting bottlenecks is that the number of NK cells infiltrated into tumor tissues is small and the activity is low.

The NK cell killing activity of 60% of NK cells on tumor cells, calculated according to the effective target ratio of the NK cells to the tumor cells of usually 25: 1, 2500 NK cells attack 100 tumor cells and only 60 tumor cells are killed, the number of tumor cells in human tumor bodies is far more than the number of NK cells, therefore, the number of NK cells and the killing activity in vivo tumor tissues are far insufficient, even if NK cells are infused intravenously, the number is limited and cannot enter tumor bodies through capillaries, which is the main reason that the curative effect of the currently used NK cell treatment method on solid tumors is not ideal, NK cells need to exert the killing activity through chemotactic action through capillaries to enter tumor body tissues through chemotactic action to compensate for the deficiency of the ratio of NK cells to tumor cells, even if NK cells are infused intravenously, the number is limited, the NK cells are not capable of entering tumor bodies through capillaries, the capillary vessels, the killing activity is enhanced, based on the principle, the invention, related genes of NK92 cells are modified through gene editing technology, NK cells overexpress chemokine receptors receptor CXCR2 and interleukin-2 (IL2) are allowed to excessively express the chemokine receptors receptor CXCR2 and interleukin 2 (IL- ×) through a CXCR × -27 cell proliferation assay after the in vitro chemotactic cell culture medium is added, the extracellular cell proliferation of a polyclonal cell culture medium, the wild cell proliferation assay, the cell proliferation is further, the extracellular chemotactic cell proliferation of CXCR-53 cell proliferation is increased, the wild cell proliferation assay, the polyclonal cell proliferation of CXCR-CD-19 cell proliferation assay, the polyclonal cell proliferation is increased, the polyclonal cell proliferation of CXCR2 cell proliferation is increased, the polyclonal cell proliferation assay is further the polyclonal cell proliferation assay is performed after the polyclonal cell proliferation assay, the polyclonal cell proliferation of the polyclonal cell proliferation assay is performed after the polyclonal cell line is performed under the polyclonal cell line of the polyclonal cell line.

In addition, the distribution of NK92 cells in SCID tumor-bearing mice injected with green fluorescent protein in a tracking way is verified by an in-animal fluorescence imaging system that genetically modified NK92 cells (LW649 NK92 cells) are successfully positioned and chemotactic to tumor bodies of subcutaneous colon cancer of mice, and compared with SCID tumor-bearing mice treated by common NK92 cells, the green fluorescence distribution area of a tumor area is increased and the intensity is obviously increased.

The conventional gene transfection can only be transiently overexpressed and has the limitation of MHC, and the transfected gene is easily lost after cell proliferation, so that NK cells can not be subjected to persistent high/overexpression of a certain protein molecule; according to the invention, the NK cell genome is edited, and related genes can be stably transmitted to progeny NK cells, so that the high expression of CXCR2 and IL2 is constant and lasting, and the NK92 cells which are chemotactic for tumor bodies and can be continuously proliferated are obtained.

In the invention, corresponding viruses are obtained by lentivirus packaging infection, cells with the expression purity of more than 80-90% are obtained by infection and integration for 48 hours, fluorescence microscope observation and puromycin resistance screening, and qPCR and ELISA detection of various groups of NK92 cells prove that chemokine receptor CXCR2 and cytokine areIL2 was successfully overexpressed in NK92 cells, and IL2 was secreted extracellularly, thus demonstrating that the present invention successfully constructs NK92-CXCR2-IL2 cell line. The qPCR detection procedure for NK92 cells was as follows: taking the cells of an experimental group (CXCR2-IL2-NK92 cells) and a control group (wild type NK92 cells) in the logarithmic growth phase to crack NK cells by a Trizol method, extracting RNA, reverse transcribing the RNA into cDNA by a Takara kit, quantitatively detecting CXCR2 and IL2 gene expression levels by qPCR fluorescence, and setting 3 multiple holes in each group. The steps of ELISA detection of IL2 secretion of NK92 cells in vitro are as follows: taking an experimental group (CXCR2-IL2-NK92 cells) and a control group (wild type NK92 cells) in a logarithmic growth phase, counting 10^7 cells, fixing the volume of a T25 culture bottle to 5ml, placing the culture bottle at 37 ℃, and adding 5% CO₂After culturing for 48 hours in an incubator, centrifuging and taking the supernatant, additionally arranging a blank medium group (negative control group), detecting the expression quantity of the IL2 protein by referring to an ELISA kit protocol, and arranging 3 more holes in each group.

The NK92-CXCR2-IL2 cell strain constructed in the research can keep the continuous and stable over-expression of CXCR2 and IL2, wherein the cytokine released by IL2 in culture supernatant can act on cells and play a role in promoting proliferation. In the invention, the proliferation function of the cells is further verified by using a CCK8 method and a test lasting for 4 weeks in mice, and the NK92 cells with over-expression of IL2 can be observed to grow and maintain an active state and proliferate continuously. The NK chemotaxis and proliferation detection in the tumor body of the mouse adopts an animal fluorescence imaging system, and experimental group and blank group NK cells injected into the tumor body of the mouse carry EGFP gene (enhanced green fluorescence protein), and can display green fluorescence in the animal fluorescence imaging system so as to know the distribution, quantity and activity of the NK cells in the mouse body.

The foregoing description is only exemplary of the preferred embodiments of the disclosure and is illustrative of the principles of the technology employed. It will be appreciated by those skilled in the art that the scope of the disclosure herein is not limited to the particular combination of features described above, but also encompasses other embodiments in which any combination of the features described above or their equivalents does not depart from the spirit of the disclosure. For example, the above features and (but not limited to) the features disclosed in this disclosure having similar functions are replaced with each other to form the technical solution.

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Claims

1. A method of making a genetically modified NK cell, the method comprising: NK cells are enabled to highly express CXCR2 and IL2 by means of genetic engineering.

2. The method of claim 1, wherein: the genetic engineering means is realized by a transcription activated CRISPR-CAS9 system.

3. The method of claim 2, wherein: in the CRISPR-CAS9 system, sgRNA target sequences for CXCR2 are: GCGAGTTCAGACAAGTCCGT, as shown in SEQ ID NO. 1; the sgRNA target sequence for IL2 was: GTGGGCTAATGTAACAAAGA, as shown in SEQ ID NO. 3.

4. The method of claim 3, wherein: the genetic engineering operation comprises the following steps: (1) construction of sgRNA vector: constructing a lentiviral vector comprising the sgRNA target sequence for CXCR2 and the sgRNA target sequence for IL 2; (2) construction of dCas9 plasmid: synthesizing a dCas9 plasmid, wherein the dCas9 plasmid has a nuclear localization signal, the C-terminal of the dCas9 plasmid has a VP64 transcription activation domain, the dCas9 plasmid has double mutations of D10A and H840A, and the dCas9 plasmid has no enzyme cutting activity; (3) infection of NK cells: transfecting 293T cells with the sgRNA vector and the dCas9 plasmid to prepare lentivirus solution, and infecting NK cells by combining virus stock after concentrating PEG8000 to obtain the genetically modified NK cells.

5. The method of claim 4, wherein: the transfection of 293T cells was performed by liposome-mediated transfection, and infection of NK cells was performed by lentivirus infection.

6. A genetically modified NK cell prepared by the preparation method of any one of claims 1-5.

7. A genetically modified NK cell that overexpresses CXCR2 and IL 2.

8. An sgRNA applied to a transcription-activated CRISPR-CAS9 system, a target sequence of the sgRNA comprising: sgRNA target sequence for CXCR2 and sgRNA target sequence for IL2, sgRNA target sequence for CXCR2 is: GCGAGTTCAGACAAGTCCGT, as shown in SEQ ID NO. 1; the sgRNA target sequence for IL2 was: GTGGGCTAATGTAACAAAGA, as shown in SEQ ID NO. 3.

9. A kit containing the sgRNA of claim 8.

10. Use of the genetically modified NK cell of claim 6 or 7 for the prevention, inhibition or treatment of a tumor.