Silencer fragment for inhibiting proliferation of K562 cells, its inhibition method and application

Abstract

The present invention discloses a silencer fragment that inhibits the proliferation of K562 cells, and the nucleotide sequence of the silencer fragment is shown as SEQ ID NO: 1-3. By knocking out these three silencers, the proliferation of K562 cells can be effectively inhibited. By knocking out the three silencers Ps1, Ps2, and Ps3, the proliferation of K562 cells can be highly inhibited, and the speed of its proliferation is an important factor directly affecting the development of leukemia. Therefore, inhibiting the proliferation of K562 cells can slow down the development of leukemia, providing a new strategy for the treatment of leukemia. It can also be used in the preparation of drugs or products for the treatment or diagnosis of leukemia, providing new methods for the diagnosis and treatment of leukemia. The obtained K562 cell line with the three silencers Ps1, Ps2, and Ps3 knocked out can be used as new materials for studying leukemia, providing new materials and research directions for the prevention or treatment of leukemia.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, and particularly to a silencer fragment for inhibiting the proliferation of K562 cells, its inhibition method and application. Background Art

[0002] Leukemia is a malignant clonal disease originating from hematopoietic stem cells. Its complex pathogenesis is associated with multiple factors such as biology, physics, chemistry, genetics, and other blood diseases. Among them, the uncontrolled proliferation of leukemia cells in the bone marrow and other hematopoietic tissues leads to the inhibition of normal hematopoiesis and infiltration into other organs. In China, the incidence of leukemia is 3 - 5 per 100,000 people, among which acute myeloid leukemia is the most common. And the incidence populations of acute leukemia and chronic leukemia are different: acute myeloid leukemia is more common in adults, while acute lymphoblastic leukemia is more common in children. There are various treatment methods for leukemia, including molecular targeted drug therapy, chemotherapy, radiotherapy, blood or bone marrow transplantation, and adjuvant therapies for various symptoms. The prognosis after treatment varies from person to person. For some patients, long-term survival or cure can be achieved through appropriate treatment, while for some patients, the disease may relapse or progress continuously. In terms of prevention, since the specific pathogenesis has not been clarified, there are currently no specific preventive measures, and only limited prevention can be achieved by avoiding exposure to known risk factors.

[0003] K562 cells (human chronic myeloid leukemia cells) are a leukemia cell line. The rate of cell proliferation is an important factor directly affecting the development of the disease. Therefore, inhibiting the proliferation of K562 cells can slow down the development of leukemia and provide a new strategy for the treatment and prevention of leukemia.

[0004] A silencer is a cis-regulatory element located in the non-coding region of the genome and plays an important role in regulating gene expression. Silencers generally inhibit gene expression by binding to inhibitory transcription factors (TFs) or changing chromatin structure. Currently, the research on silencers mainly focuses on the mechanism level, and less on their biological functions. And there is no report on the research of silencers in the field of K562 cell proliferation or the treatment / prevention of leukemia. Summary of the Invention

[0005] The purpose of the present invention is to provide a silencer fragment for inhibiting the proliferation of K562 cells, its inhibition method and application, so as to provide a new idea for the prevention and treatment of leukemia by inhibiting the proliferation of K562 cells.

[0006] According to the first aspect of the present invention, a silencer fragment for inhibiting the proliferation of K562 cells is provided. The nucleotide sequence of the silencer fragment is shown as SEQ ID NO: 1 - 3. Thus, by knocking out these three silencer fragments, the proliferation of K562 cells can be inhibited, or it can be used as a new approach for the prevention and treatment of leukemia.

[0007] According to a second aspect of the present invention, there is provided the use of a silencer fragment in inhibiting the proliferation of K562 cells, and the nucleotide sequence of the silencer fragment is as shown in SEQ ID NO: 1-3. Thus, by efficiently inhibiting the proliferation of K562 cells, new ideas and methods can be provided for the research, diagnosis, prevention and treatment of leukemia.

[0008] According to a third aspect of the present invention, there is provided the use of a silencer fragment in the preparation of a drug for preventing and treating leukemia. Thus, new solutions can be provided for the prevention and treatment of leukemia.

[0009] According to a fourth aspect of the present invention, there is provided a method for inhibiting the proliferation of K562 cells, which is achieved by simultaneously knocking out three silencers in K562 cells, and the three silencers are located at positions 87964523-87964773 bp, 87915951-87916201 bp, and 87953529-87954017 bp on chromosome 10; the nucleotide sequences of the three silencers are as shown in SEQ ID NO: 1-3. Thus, by this method, the proliferation of K562 cells can be efficiently inhibited, providing new strategies and methods for the study of leukemia.

[0010] In some embodiments, the method comprises the following steps: S1: Design and synthesize the sgRNA sequences for knockout according to the nucleotide sequences of the three silencers; S2: Connect the synthesized sgRNA sequences to the PX459 V2 vector; S3: Transfect the plasmid obtained by connection in step S2 into K562 cells, and the three silencers in K562 cells can be knocked out.

[0011] In some embodiments, the sgRNA sequences are as shown in SEQ ID NO: 4-9.

[0012] According to a fifth aspect of the present invention, there is provided the use of the above method in inhibiting the proliferation of K562 cells. Thus, by this method, the proliferation of K562 cells can be efficiently inhibited, developing a new way and method for the prevention, treatment and research of leukemia.

[0013] According to the sixth aspect of the present invention, a method for preparing a K562 cell line with a silencer knocked out is provided. The silencer includes any one or a combination of multiple locations at positions 87964523 - 87964773 bp, 87915951 - 87916201 bp, and 87953529 - 87954017 bp on chromosome 10. The nucleotide sequence of the silencer is as shown in SEQ ID NO: 1 - 3. The method includes the following steps: S1: Design and synthesize the sgRNA sequence for knockout according to the nucleotide sequence of the silencer, and the sgRNA sequence is as shown in SEQ ID NO: 4 - 9; S2: Connect the synthesized sgRNA sequence to the PX459 V2 vector; S3: Transfect the plasmid obtained by connection in step S2 into K562 cells; S4: Screen the cells transfected in step S3 with a medium containing puromycin; S5: Detect the surviving cells obtained by screening in step S4 to obtain positive cells with the silencer knocked out, and amplify and culture the positive cells to obtain a K562 cell line with the silencer knocked out. Thus, through this method, a K562 cell line with one or two or three silencers knocked out can be efficiently obtained, providing new materials for the research of leukemia.

[0014] According to the seventh aspect of the present invention, a new K562 cell line is provided. This K562 cell line is prepared by the above method, and three silencers on its chromosome 10 are knocked out. The three silencers are located at positions 87964523 - 87964773 bp, 87915951 - 87916201 bp, and 87953529 - 87954017 bp on chromosome 10 respectively; the nucleotide sequences of the three silencers are as shown in SEQ ID NO: 1 - 3. Thus, this new cell line can be used as new materials for the research of leukemia, providing research materials and directions for the research of new leukemia targeted drugs.

[0015] According to the eighth aspect of the present invention, the application of the described K562 cell line in the preparation of products for preventing, treating, or diagnosing leukemia is provided. Three silencers on chromosome 10 of this K562 cell line are knocked out.

[0016] Advantages of the present invention: The present invention discloses three silencer fragments, Ps1, Ps2, and Ps3, which inhibit the proliferation of K562 cells. By knocking out these three silencers, the proliferation of K562 cells can be effectively inhibited. By knocking out Ps1, Ps2, and Ps3, the proliferation of K562 cells can be highly inhibited. The rate of proliferation directly affects the development of leukemia. Therefore, inhibiting the proliferation of K562 cells can slow down the development of leukemia and provide a new strategy for the prevention and treatment of leukemia. It can also be used in the preparation of drugs or products for the treatment or diagnosis of leukemia, providing new methods for the diagnosis, prevention, and treatment of leukemia. The obtained K562 cell line with Ps1, Ps2, and Ps3 knocked out can be used as new materials for studying leukemia, providing new materials and research directions for the treatment of leukemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is the result diagram of the dual-luciferase experiment to verify the activities of the three silencers, Ps1, Ps2, and Ps3: In the figure, *** indicates extremely significant differences ( p <0.001);

[0018] Figure 2 It is the result diagram of the PCR detection after the silencer knockout: In the figure, the first lane from the left is the marker, the second lane is the electrophoresis band of the Ps1 silencer WT wild type, the third lane is the electrophoresis band after the Ps1 silencer knockout, the fourth lane is the electrophoresis band of the Ps2 silencer WT wild type, the fifth lane is the electrophoresis band after the Ps2 silencer knockout, the sixth lane is the electrophoresis band of the Ps3 silencer WT wild type, and the seventh lane is the electrophoresis band after the Ps3 silencer knockout;

[0019] Figure 3 It is the result diagram of the detection of the proliferation of K562 cells and wild-type cells after knocking out all three silencers, Ps1, Ps2, and Ps3: Among them, WT on the left figure represents wild-type K562 cells, and Ps1_Ps2_Ps3-KO on the right figure represents K562 cells with all three silencers, Ps1, Ps2, and Ps3, knocked out;

[0020] Figure 4 It is the result diagram of the CCK8 detection of cell proliferation: Among them, WT represents the detection result of wild-type K562 cells, and Silencers KO represents the detection result of K562 cells with all three silencers, Ps1, Ps2, and Ps3, knocked out. In the figure, ** indicates extremely significant differences ( p <0.01). DETAILED DESCRIPTION OF THE EMBODIMENTS

[0021] The present invention will be further described in detail below with reference to the drawings and embodiments.

[0022] Example 1: Screening of silencers

[0023] The target silencer was screened using the silencer screening system and its screening method disclosed in Patent CN112538493A. The specific steps are as follows:

[0024] After culturing K562 cells, the K562 cells were collected and genomic DNA was extracted, which was fragmented into DNA fragments of about 300 bp. The fragmented DNA fragments were ligated to Illumina adapters, and PCR amplification was performed using primers containing the homologous arm sequences of the MAS-seq vector. The PCR products were purified, and then the purified PCR products were recombinated homologously into the MAS-seq vector to construct a MAS-seq screening plasmid. The MAS-seq screening plasmid was transfected into 293T cells, RNA was extracted, and the mRNA of the marker gene was enriched. The mRNA of the marker gene was reverse-transcribed to obtain cDNA, and the cDNA was PCR amplified. The PCR products were purified, and the purified PCR amplification products (output library) were subjected to high-throughput sequencing;

[0025] The blank MAS-seq vector without any screening fragments was transfected into 293T cells, and total RNA was extracted. The mRNA of the marker gene with a poly (A) tail was isolated and purified, and reverse-transcribed into cDNA. The reverse-transcription product was treated with RNase A and RNaseH to obtain purified cDNA. The obtained cDNA was PCR amplified, and the purified PCR products (input library) were subjected to high-throughput sequencing.

[0026] Then, through the analysis of the sequencing data of the input and output libraries by the CRADLE software, three silencers were found on chromosome 10, denoted as Ps1, Ps2, and Ps3 respectively. Their specific positions in the genome (hg38 reference genome) are shown in Table 1:

[0027] Table 1 Positions and sequences of the three silencers Ps1, Ps2, and Ps3

[0028] Silencer name Genomic location Nucleotide sequence Ps1 Chr10: 87964523-87964773 As shown in SEQ ID NO:1 Ps2 Chr10: 87915951-87916201 As shown in SEQ ID NO:2 Ps3 Chr10: 87953529-87954017 As shown in SEQ ID NO:3

[0029] The nucleotide sequences of the above three silencers Ps1, Ps2, and Ps3 are as follows:

[0030] Ps1 silencer (SEQ ID NO:1):

[0031] CCCTGTCTCAAAAACACACACACACACACACACACACACAAAGAAATACATTGATTTTTCACATAGGTAGTAAGAGAAACATTCTTTTTGAACTCAGCTGTTTGTGAATTGAATTTTGTAATTCAAATGCTATATTATGTAAACTATTGATGACTTTCAATCTGCATTTATTTTGTATAATTATTTAGTTAATATTTGCCACTTATATTCCTTAAAAAATAAAATTGAGGTTGGGCGTGGTGGCTCACACT

[0032] Ps2 silencer (SEQ ID NO:2):

[0033] GATGTTTATATATTGAAATTATTGGAAGTAAGGTATGTTTATATTAGAAAGATTTGTAGTCTAGATTATCCAAGTTTTGGGAGTATTACCTCTCTGCTTTTGTTTATCTACTTTTTTAGTCTCTACTTTCCAAGTATCTATAGGCAAATTTTCCCATTTCCCTTTGGAAAGTGCTGTTTTCTTGCTTTTTTTCCGCCTTTCCATTGTGTCAGACTTATAAGGCAATCAGCCAACTGTGGGCATGAAATCCT

[0034] Ps3 silencer (SEQ ID NO:3):

[0035] TCGCTTGATCTCAGGAGTTCGAGACCAGCCTGGACAACAAGGTGAAACCCCATCTCTACTAAAAACACAAAAATTAGCTGGGCATAGTGGCACACACCTGTAGTCCCAGCTTCTTGGGAGGCTGAGGCAGGAGAATCGCTTGAATCCAGGAGGCAGAGGTTGCAGTTAGCCGAGATCATGCCACTGCACTGCAGCCTGGACATCGGAGCAAGACTTTGTCTTAGAAATAAATAAATAAATATAAAATAAAATAAATGGGAAGTTGTGTATATAAATTATAAATGCTACATTCAGAAAAGCTTTTGAAGGTTGTCAGACAGTTTCTTAAAGGAAGTTCACCAGTTCTTTATTGAACATTGAAGAAAACATACAGTTTAGACTGGCATTAAAACTGAAAGAAGTGGCCAGACGCAGTGGTAGACGCAGTGGTTCACGCCTGTAATCCCAGCACTTTGGGAGGTCAAGGTGGATGGATCACCTGAGGTCAGG

[0036] Example 2 Verification of silencer activity

[0037] First, the homologous arm sequences left: AACATTTCTCTGGCCTAACTGGCCG (SEQ ID NO:16); right: CGTACCTGAGTCTTCCACGGGGTTGG (SEQ ID NO:17) were added to both ends of the 3 silencers, and the silencer detection sequences with homologous arm sequences were artificially synthesized.

[0038] Using homologous recombinase (Vazyme, #C117), the silencer with homologous arm sequences was cloned upstream of the PGK promoter of vector PGL4.53 (Promega, #E1960). The constructed plasmid was co-transfected with the pRL-CMV Renilla reporter vector (Promega, #E1960) into cells. After 24 hours of transfection, the cells were treated with luciferase lysis buffer to prepare cell lysates. The cell lysates were added to 96-well plates, and the dual-luciferase activity was detected according to the following steps:

[0039] Firefly luciferase activity detection: Add the firefly luciferase substrate and immediately read the luminescence intensity of the firefly luciferase.

[0040] Renilla luciferase activity detection: Add Renilla luciferase substrate and read the luminescence intensity of Renilla luciferase.

[0041] Calculate the ratio of firefly luciferase signal to Renilla luciferase signal as the relative activity value of this experimental group.

[0042] The detection results are as Figure 1 shown. Compared with the control group (PGL4.53), the detection values of silencers Ps1, Ps2, and Ps3 were extremely significantly decreased ( p <0.001), indicating that Ps1, Ps2, and Ps3 have strong silencer activity and can be used for subsequent research.

[0043] Example 3 Knockout and identification of three silencers Ps1, Ps2, and Ps3

[0044] Design sgRNAs for knocking out three silencers. The specific sequences are as follows:

[0045] Ps1 F sgRNA (SEQ ID NO:4): GGACTGTTAGTGTGTAGTACAGG,

[0046] Ps1 R sgRNA (SEQ ID NO:5): GGTGACTCACTGCTACTTTACGG;

[0047] Ps2 F sgRNA (SEQ ID NO:6): GGTCCATAGTGAAGCGTTTGAGG,

[0048] Ps2 R sgRNA (SEQ ID NO:7): GGAAGCTTATCTGTCAAGCTTGG;

[0049] Ps3 F sgRNA (SEQ ID NO:8): GGAGGTGGTTACAGGGCTCGTGG,

[0050] Ps3 R sgRNA (SEQ ID NO:9): GGCAGGCTTTGTAGGCCACAGGG.

[0051] Knockout of three silencers, namely Ps1, Ps2, and Ps3, in K562 cells by CRISPR / Cas9 technology: The CRISPR / Cas9 expression vector PX459 V2 (Addgene, #118632) was digested with the restriction endonuclease BbsI, and then these sgRNAs (Ps1 F sgRNA, Ps1 R sgRNA, Ps2 F sgRNA, Ps2 R sgRNA, Ps3 F sgRNA, Ps3 R sgRNA) were ligated to the linearized vector PX459 V2 using T4 DNA ligase respectively. In this way, the PX459 V2 vectors containing 6 different sgRNAs were obtained. Then, these 6 vector plasmids were mixed together and their concentrations were measured. K562 cells were cultured and transfected into K562 cells by electroporation. The transfection condition was: 1 μg plasmid / million cells. After 24 hours of transfection, the K562 cell medium was replaced with a selection medium containing 1.5 μg / mL puromycin. During the selection process, untransfected cells would die quickly due to puromycin inhibiting protein synthesis and usually detached within 24 - 72 hours. Transfected cells carrying the PuroR gene could survive due to their resistance to the toxicity of puromycin.

[0052] After 3 days of screening the transfected cells with the selection medium containing puromycin, the normal medium (without puromycin) was replaced. Then, the surviving K562 cells were sorted by flow cytometry to obtain single cells. Next, the sorted single cells were placed in a 96 - well plate for expansion culture (one cell per well). After the cells grew confluent, they were transferred to a 24 - well plate and continued to be cultured until the confluence was over 80%. Half of the cells were digested and continued to be cultured for subsequent research, and the other half was used for PCR detection. The principle of PCR detection is: If the silencer is successfully knocked out, then corresponding primers are designed to amplify the wild - type and the knocked - out positive cell lines. The length of the PCR amplification product of the positive cell line will be shorter than that of the wild - type. For example, if PS1 is successfully knocked out, the length of the PCR product of wild - type cells is 620 bp, and the length of the PCR product of the knocked - out positive cell line is 369 bp.

[0053] The detection primers for each silencer are respectively:

[0054] Ps1 primer F (SEQ ID NO:10): TCTCCAGGTCGATTTGACCA,

[0055] Ps1 primer R (SEQ ID NO:11): GAGTCGTTGCTCACCTTTGC;

[0056] Ps2 primer F (SEQ ID NO:12): TGACTGAATCGTGTTGACCC,

[0057] Ps2 primer R (SEQ ID NO:13): TGACTGAATCGTGTTGACCC;

[0058] Ps3 primer F (SEQ ID NO:14): CTGTGGTGCTGTCATCCTTT,

[0059] Ps3 primer R (SEQ ID NO:15): ACTCTGCCTCAAGGATGACA.

[0060] After extracting DNA from each sample cell, simultaneously amplify it with these three sets of primers. When the detection results of PCR amplification with all three sets of primers are positive, the cell is a cell with all three silencers knocked out, and the remaining half of the cells of this cell are retained for continued amplification culture. For example, when the detection results of the PCR amplification products of this cell are all as Figure 2 shown, it indicates that all three silencers of this cell have been knocked out, specifically as follows: The PCR amplification product of Ps1 KO is shorter than that of the left control group WT, indicating that the silencer Ps1 has been knocked out; the PCR amplification product of Ps2 KO is also shorter than that of the left control group WT, indicating that the silencer Ps2 has also been knocked out; the PCR amplification product of Ps3 KO is also shorter than that of the left control group WT, indicating that the silencer Ps3 has also been knocked out; therefore, all three silencers Ps1, Ps2, and Ps3 of this cell have been knocked out. After PCR electrophoresis detection, after the K562 cells with all three silencers Ps1, Ps2, and Ps3 knocked out are amplified and cultured, some are taken for gene sequencing, and the sequencing results further indicate that all three silencers Ps1, Ps2, and Ps3 have been knocked out. After the K562 cells with all three silencers Ps1, Ps2, and Ps3 knocked out are amplified and cultured, a K562 cell line with all three silencers Ps1, Ps2, and Ps3 knocked out can be obtained, and this cell line can also be used as a new material for leukemia research.

[0061] Example 4 Research on the phenotypic changes of K562 cells after knocking out all three silencers Ps1, Ps2, and Ps3

[0062] Detect the proliferation of K562 cells after knocking out all three silencers Ps1, Ps2, and Ps3. First, inoculate the same number of K562 cells after knocking out all three silencers Ps1, Ps2, and Ps3 and WT wild-type cells (WT wild-type cells refer to K562 cells that have not been treated with anything) in a 100 mm culture dish, and the number of inoculated cells is 2×10 6, after 48 hours, an obvious difference in cell quantity was observed, and the results are as Figure 3 shown in Figure 3 In the right figure of Figure 3 , Ps1_Ps2_Ps3-KO represents K562 cells with all three silencers Ps1, Ps2, and Ps3 knocked out. The cell quantity is significantly less than that of the WT wild-type cells in the left figure, indicating that the proliferation rate of K562 cells with all three silencers Ps1, Ps2, and Ps3 knocked out is significantly slower than that of the wild-type group, suggesting that knocking out all three silencers Ps1, Ps2, and Ps3 can effectively inhibit the proliferation of K562 cells.

[0063] In addition, the CCK8 kit (Beyotime, C0041) was also used to detect the cell proliferation situation, and the results are as Figure 4 shown in Figure 4 : Silencers KO represents K562 cells with all three silencers Ps1, Ps2, and Ps3 knocked out. The CCK8 OD value is extremely significantly lower than that of the WT wild-type cells, indicating that the proliferation rate of K562 cells with all three silencers Ps1, Ps2, and Ps3 knocked out is significantly slower than that of the wild-type, suggesting that knocking out all three silencers Ps1, Ps2, and Ps3 can effectively inhibit the proliferation of K562 cells.

[0064] In summary, simultaneously knocking out the three silencers Ps1, Ps2, and Ps3 in K562 cells can effectively inhibit the proliferation of K562 cells, and the speed of its proliferation is an important factor directly affecting the development of leukemia. Therefore, the proliferation of K562 cells can be inhibited by knocking out the three silencers Ps1, Ps2, and Ps3 in K562 cells, thereby slowing down the development of leukemia. For example, sgRNAs (such as SEQ ID NO:4 - 9) used to knock out the three silencers Ps1, Ps2, and Ps3 can be paired with a knockout vector (such as a CRISPR / Cas9 expression vector) to prepare corresponding drugs for the treatment of leukemia; or siRNAs that can inhibit the functions of the three silencers Ps1, Ps2, and Ps3 can be made into corresponding drugs, which can also be used for the prevention and treatment of leukemia; or any method that can knock out the three silencers Ps1, Ps2, and Ps3 can be used to inhibit the proliferation of K562 cells and then for the treatment of leukemia. The obtained K562 cell line with all three silencers Ps1, Ps2, and Ps3 knocked out can be used as a new material for leukemia prevention and treatment research, providing new strategies for the research, prevention, and treatment of leukemia. Therefore, knocking out the three silencers Ps1, Ps2, and Ps3 in K562 cells can effectively inhibit their proliferation, providing new ideas and directions for the research and treatment of leukemia.

Claims

1. A method for inhibiting the proliferation of K562 cells, wherein, The method is achieved by simultaneously knocking out three silencers in K562 cells. The three silencers are located at positions 87964523 - 87964773 bp, 87915951 - 87916201 bp, and 87953529 - 87954017 bp on chromosome 10 respectively. The nucleotide sequences of the three silencers are as shown in SEQ ID NO:1 - 3.

2. According to the method described in claim 1, wherein, The method includes the following steps: S1: Design and synthesize the sgRNA sequences for knockout according to the nucleotide sequences of the three silencers. S2: ligate the synthesized sgRNA sequences to the PX459 V2 vector. S3: Transfect the plasmid obtained by ligation in step S2 into K562 cells, and then the three silencers in K562 cells can be knocked out.

3. The method according to claim 2, wherein The sgRNA sequences are as shown in SEQ ID NO:4 - 9.

4. Application of the method according to claim 2 or 3 in inhibiting the proliferation of K562 cells.

5. Preparation method of K562 cell line with silencer knocked out, wherein, The silencers are three silencers located at positions 87964523 - 87964773 bp, 87915951 - 87916201 bp, and 87953529 - 87954017 bp on chromosome 10 respectively. The nucleotide sequences of the three silencers are as shown in SEQ ID NO:1 - 3. The method includes the following steps: S1: Design and synthesize the sgRNA sequences for knockout according to the nucleotide sequences of the silencers. The sgRNA sequences are as shown in SEQ ID NO:4 - 9. S2: ligate the synthesized sgRNA sequences to the PX459 V2 vector. S3: Transfect the plasmid obtained by ligation in step S2 into K562 cells. S4: Screen the cells transfected in step S3 with a medium containing puromycin. S5: Detect the surviving cells obtained by screening in step S4 to obtain positive cells with silencer knockout, and amplify and culture the positive cells to obtain a K562 cell line with three silencers simultaneously knocked out.

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