CN112481305A

CN112481305A - Method for constructing EPB41 gene knockout THP-1 cell line based on CRISPR-Cas9 system

Info

Publication number: CN112481305A
Application number: CN202011374637.9A
Authority: CN
Inventors: 乔凯旋; 郑永唐; 刘鑫; 刘丰亮; 范雪刚
Original assignee: Zhengzhou University; Kunming Institute of Zoology of CAS
Current assignee: Zhengzhou University; Kunming Institute of Zoology of CAS
Priority date: 2020-11-30
Filing date: 2020-11-30
Publication date: 2021-03-12

Abstract

The invention discloses a method for constructing an EPB41 gene knockout THP-1 cell line based on a CRISPR-Cas9 system. The method takes the protein coding sequence in the first exon, the protein coding sequence in the fourth exon and the protein coding sequence in the seventh exon of the human EPB41 gene, and the ' N18 or N20 ' of the fragment which accords with the sequence rule arrangement of 5 ' -G-N18-NGG-3 or 5 ' -G-N20-NGG-3 ' or 5 ' -CCN-N18-C-3 ' or 5 ' -CCN-N20-C-3 ' as the target sequence; the THP-1 cell line with the EPB41 gene knocked out stably is obtained and transmitted to the next generation along with the division and the proliferation of the cells. Experiments prove that the THP-1 cell line with the EPB41 gene knocked out, which is obtained by the invention, cannot correctly express the 4.1R protein. The research on the influence of the deletion of the protein 4.1R on the functions of cell membranes can also be used for researching the direct or indirect interaction of the protein 4.1R and certain immunoreaction or other signal pathway protein molecules.

Description

Method for constructing EPB41 gene knockout THP-1 cell line based on CRISPR-Cas9 system

The technical field is as follows:

the invention relates to a THP-1 cell line for knocking out EPB41 gene constructed based on a CRISPR-Cas9 system, belonging to the technical field of biology.

Background

THP-1 is a human peripheral blood mononuclear cell line, originally derived from acute monocytic leukemia patients, belonging to the group of suspension cells, suitable for transfection or infection experiments, and having HLA types as surface antigens: a2, a9, B5, DRw1, DRw 2. Protein 4.1R (EPB41 gene) is a membrane-cytoskeletal cross-linking agent and adaptor that binds cytoplasmic fibrin filament complexes and a variety of transmembrane proteins. In addition, protein 4.1R is present in CD4⁺The initial stages of T lymphocyte activation and signal transduction exert negative regulatory effects. Protein 4.1R by targeting CD8⁺Negative regulation of the levels of LAT and ERK protein phosphorylation in T cells thereby negatively regulating CD8⁺T cell activation and proliferation. Protein 4.1R by inhibition of CD4⁺T cell activation thereby mitigates autoimmune pathological damage in the mouse encephalomyelitis (EAE) model induced by the mouse autoantigen MOG.

In view of the important role of the protein 4.1R in immune response, the cell line with the EPB41 gene knocked out is important for researching the function of the protein 4.1R, and the 4.1R-deficient mice have the problems of low fertility rate, long breeding period and the like. The human 4.1R deleted cell line is not reported, and in order to better research the function of 4.1R, particularly the function in some immune reactions, the invention of a cell line for knocking out EPB41 gene is urgently needed to be invented for the research of the function of 4.1R, including the research of the influence of the deletion of the protein 4.1R on the function of cell membranes, and the research of the direct or indirect interaction of the protein 4.1R and some immune reactions or other signal path protein molecules.

Disclosure of Invention

The invention aims to provide a cell line for knocking out a human EPB41 gene based on a CRISPR-Cas9 editing technology and a construction method thereof.

A method for constructing an EPB41 gene knockout cell line based on a CRISPR-Cas9 system takes a protein coding sequence in a first exon, a protein coding sequence in a fourth exon and a protein coding sequence in a fifth exon of a human EPB41 gene, and ' N18 or N20 ' of a fragment which accords with the sequence regular arrangement of 5 ' -G-N18-NGG-3 or 5 ' -G-N20-NGG-3 ' or 5 ' -CCN-N18-C-3 ' or 5 ' -CCN-N20-C-3 ' as a target sequence; n represents any one of A, T, C, G, where "N18" and "N20" are 18 and 20 deoxynucleotides, respectively.

Preferably, the target sequences are as shown in Seq-No.1, Seq-No.2 and Seq-No. 3.

Preferably, the sequence of the target sequence Seq-No.1 is located in the first exon sequence of the EPB41 gene; the sequence of Seq-No.2 is located in the 4 th exon sequence of EPB41 gene; the sequence of Seq-No.3 is located in the 7 th exon sequence of the EPB41 gene.

Depending on the target sequence, the method is method A or method B or method C as follows:

method A (target sequence SEQ ID No.1) comprising the steps of:

(a1) design of sgRNA and Synthesis of oligonucleotide chain, and Synthesis of oligonucleotide chain 1 sgRNA was designed based on exon I, exon IV and exon seventh of human EPB41 gene, and 2 single-stranded DNAs named forward single-stranded DNA-1 and reverse single-stranded DNA-1 were synthesized, the sequence of the forward single-stranded DNA-1 being, for example, the sequence of reverse single-stranded DNA-1 described in Seq-No.4, such as Seq-No. 5;

(a2) carrying out annealing reaction on the forward single-stranded DNA-1 and the reverse single-stranded DNA-1 to obtain double-stranded DNA-1;

(a3) connecting the double-stranded DNA-1 to a cutting site of a restriction enzyme BsmBI of a LentiCRISPR V2 plasmid to obtain a recombinant plasmid which is marked as LentiCRISPR V2(B) -gRNA-EPB 41-1;

(a4) the LentiCRISPR V2(B) -gRNA-EPB41-1, pCMV-VSV-G and psPAX2 plasmids are co-transfected into 293T cells to wrap out lentivirus supernatant;

(a5) adding the lentivirus supernatant into THP-1 cells, and screening by puromycin to obtain an EPB41 gene knockout cell line;

method B (target sequence SEQ ID No.2) comprising the steps of:

(b1) designing sgRNA and synthesizing oligonucleotide chains, and synthesizing 1 sgRNA according to the fourth exon of the human EPB41 gene to synthesize 2 single-stranded DNAs named as forward single-stranded DNA-2 and reverse single-stranded DNA-2, wherein the forward single-stranded DNA-2 has a sequence shown as Seq-No. 6; the sequence of the reverse single-stranded DNA-2 such as Seq-No.7(b2) causes the forward single-stranded DNA-2 and the reverse single-stranded DNA-2 to carry out annealing reaction to obtain double-stranded DNA-2;

(b3) connecting the double-stranded DNA-2 to a cutting site of a restriction enzyme BsmBI of a LentiCRISPR V2 plasmid to obtain a recombinant plasmid which is marked as LentiCRISPR V2(B) -gRNA-EPB 41-2;

(b4) the LentiCRISPR V2(B) -gRNA-EPB41-2, pCMV-VSV-G and psPAX2 plasmids are co-transfected into 293T cells to wrap out lentivirus supernatant;

(b5) adding the lentivirus supernatant into THP-1 cells, and screening by puromycin to obtain an EPB41 gene knockout cell line;

method C (target sequence SEQ ID No.3), comprising the steps of:

(c1) design of sgRNA and synthesis of oligonucleotide chain, and synthesis of oligonucleotide chain 1 sgRNA is designed according to the seventh exon of human EPB41 gene, and 2 single-stranded DNAs named as forward single-stranded DNA-3 and reverse single-stranded DNA-3 are synthesized, wherein the sequence of the forward single-stranded DNA-3 is shown as Seq-No. 8; the reverse single-stranded DNA-3 sequence such as Seq-No.9(c2) annealing the forward single-stranded DNA-3 and the reverse single-stranded DNA-3 to obtain a double-stranded DNA-3;

(c3) connecting the double-stranded DNA-2 to a cutting site of a restriction enzyme BsmBI of a LentiCRISPR V2 plasmid to obtain a recombinant plasmid which is marked as LentiCRISPR V2(B) -gRNA-EPB 41-3;

(c4) the LentiCRISPR V2(B) -gRNA-EPB41-3, pCMV-VSV-G and psPAX2 plasmids are co-transfected into 293T cells to wrap out lentivirus supernatant;

(c5) and adding the lentivirus supernatant into THP-1 cells, and screening by puromycin to obtain an EPB41 gene knockout cell line.

Further, in the step (a3), the step (B3) and the step (c3), the plasmids are LentiCRISPR V2(B) -gRNA-EPB41-1, LentiCRISPR V2(B) -gRNA-EPB41-2 and LentiCRISPR V2(B) -gRNA-EPB41-3, respectively.

The second objective of the present invention is to protect the cell lines HP-1-KO-EPB41-1, THP-1-KO-EPB41-2 and THP-1-KO-EPB41-3 with the EPB41 gene knocked out, which are prepared by the above method, and the applications thereof.

The principle and the most central key technology of the invention are that sgRNA of a targeted human EPB41 gene is scientifically and reasonably constructed, then the sgRNA is cloned to lentiCRISPR v2 plasmid with Cas9 gene and the plasmid is transfected into cells, gene silencing is achieved by using a CRISPR-Cas9 system, and a cell line knocked out by human EPB41 is obtained through drug screening.

The invention has the advantages that:

1, THP-1 cell line with EPB41 gene knocked out, can not express FSCN1 protein correctly due to deletion frame shift mutation of some fragments after single site cutting.

2, the invention designs three sgRNAs aiming at the human cell line for the first time.

3, the EPB41 gene knockout constructed by the invention forms frame shift mutation on the genome level, so the EPB41 gene knockout can be transmitted to the next generation along with the division and proliferation of cells to form a stable EPB41 gene knockout cell line.

The application of the invention is as follows: 1, the cell line can be used as an experimental material for researching the function of protein 4.1R on cell membranes.

2, the cell line can be used as an experimental material for researching the direct or indirect interaction between the protein 4.1R and some protein molecules.

3, can be used for researching the influence of the deletion protein 4.1R on the phosphorylation of key signal molecules in some signal paths.

Drawings

FIG. 1 is a diagram of electrophoresis after digestion of lentiCRISPR v2 carrier plasmid, wherein about 11000bp is the objective fragment after digestion;

FIG. 2 shows the result of sequencing a ligated plasmid by ligating the primer annealing to the digested lentiCRISPR v2 vector.

FIG. 3 PCR amplification products of the knock-out cell line EPB41, g1, g2 and g3 are electrophoretograms (about 200bp) of the genomic DNA flat PCR amplification products of the cell lines THP-1-KO-EPB41-1, THP-1-KO-EPB41-2 and THP-1-KO-EPB41-3, respectively.

FIG. 4 shows the sequencing results of PCR amplification products of genomic DNAs of cell lines, in which FIG. 4(1-1), FIG. 2-2 and FIG. 3-3 show the sequencing results of PCR amplification products of genomic DNAs of THP-1-KO-EPB41-1, THP-1-KO-EPB41-2 and THP-1-KO-EPB41-3, respectively.

FIG. 5EPB41 knockout cell line and THP-1 cell line transfected with lentiCRISPR V2 plasmid not ligated to gRNA (as a positive control for subsequent experiments), in which lanes V2 are THP-1 cell lines transfected with lentiCRISPR V2 plasmid not ligated to gRNA, and lanes g1, g2 and g3 are THP-1-KO-EPB41-1, THP-1-KO-EPB41-2 and THP-1-KO-EPB41-3 cell lines, respectively.

FIG. 6 is a graph comparing the viral loads of V2 and g1 cells infected with HIV.

Detailed Description

The invention will be described in detail below with reference to the accompanying drawings.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 construction method of EPB41 gene-knocked cell line based on CRISPR-Cas9 editing technology

Design of sgRNA and synthesis of oligonucleotide chains, three sgRNAs are designed respectively aiming at No.1 exon, No.4 exon and No.7 exon of human EPB41 gene, and oligonucleotide chains are synthesized. The target sequences are shown in Seq-No.1, Seq-No.2 and Seq-No. 3.

LentiCRISPR v2 enzyme cleavage

(1) LentiCRISPRV2 digestion and end dephosphorylation:

cut at 37 ℃ for 30 min.

(2) 1% agarose gel electrophoresis detection (see FIG. 1 for results); the large LentiCRISPRV2 fragment was recovered from the TIANgel Midi Purification Kit gel and the concentration was determined.

Construction of LentiCRISPR v2 vector

(1) Annealing and phosphorylation of gRNA primers:

Oligo 1(100μM)1μL

Oligo 2(100μM)1μL

10×T4 Ligation Buffer 1μL

T4 PNK 0.5μL

ddH₂O 6.5μL

37 ℃, 30min, 95 ℃, 5 min; then decrease 5 ℃ per minute until it reaches 25 ℃.

(2)1: 200 dilution of each of the above double stranded fragments in sterile water.

(3) Connecting:

water bath at 25 deg.c for 30 min.

(4) Transformation; the fast DH 5. alpha. competent cells were thawed on ice, and 1. mu.L of the ligation product was added for 30min on ice. Plating and culturing at 37 ℃. Selecting single clone, adding 10mL of Amp liquid LB into each glass test tube, shaking the bacteria for culture and preserving the bacteria. Plasmid extraction, concentration determination and sequencing identification (see figure 2 for results).

Construction of EPB41 Gene knockout THP-1 cell line

(1) 293T cells were washed with 1 XPBS and digested with 0.25% trypsin.

(2)293T cells were counted after centrifugation at 800rpm, 4X 10⁵one/mL.

(3) 0.5mL of 10% FBS RPMI-1640 cell suspension was added to each well, and the cells were plated in 24-well plates and cultured at 37 ℃ for 24 hours. Cells are grown to 70% -90% for transfection of CRISPR plasmids.

(4) Transfection of 293T cells in 24-well plates: reference to

2000 Reagent kit instructions, Lipo20002 μ L/well. 540ng of DNA per well, wherein the target plasmid, LentiCRISPR v2 vector is 240ng, pCMV-VSV-G is 120ng and psPAX2 is 180 ng. Negative control was correspondingly transfected with LentiCRISPR v2 empty vector 240 ng/well.

(5) After 2 days of transfection, the supernatant of 293T cells-enveloped lentivirus was collected and centrifuged at 3000rpm for 20 min. Freezing the supernatant at-80 deg.C.

(6) THP-1 cells were counted, centrifuged and resuspended at 2X 10 per well⁴And the obtained product is paved in a U-shaped bottom plate with 96 holes. 100 u L/hole.

(7) 293T cell supernatant was added to THP-1 cells at 100. mu.L/well, and 293T cells were excised. Supplement 2mg/mL Polybrene 8. mu.L/well.

(8) Culturing for 2-3 days. 500mL of 10% FBS RPMI-1640 was added with 10mg/mL of uromycin 100. mu.L and used as CRISPR screening medium at 4 ℃ for later use.

(9) The 96-well U-plate was centrifuged at 300g for 5min and 200. mu.L/well of 2. mu.g/mL Puromycin 10% FBS RPMI-1640 was added.

(10)37℃、5％CO₂The cells were cultured in a cell incubator for 3 days and observed for lentivirus infection.

(11) Changing the screening culture medium every 3-4 days, continuously screening for about one month, transferring into 48-or 24-well plate for continuous amplification culture when cell mass is visible, and transferring into T₂₅cm²Culturing in a cell culture bottle.

(12) The selected THP-1-KO-EPB41-1, THP-1-KO-EPB41-2 and THP-1-KO-EPB41-3 cell lines were frozen.

Example 2 identification of Gene level of EPB41 knockout THP-1 cell line

(1) Extracting the genome DNA in the three cell lines of THP-1-KO-EPB41-1, THP-1-KO-EPB41-2 and THP-1-KO-EPB 41-3. (Tiangen genome extraction kit catalog number: DP304)

(2) Designing three pairs of identification primers aiming at the target sites of the three sgRNAs, wherein the forward identification primer sequence and the reverse identification primer sequence of a cell line THP-1-KO-EPB41-1 are respectively shown as Seq-No.10 and Seq-No. 11; the cell line THP-1-KO-EPB41-2 identifies the primer sequences in the forward and reverse directions as Seq-No.12 and Seq-No.13, respectively; the forward and reverse identification primer sequences of cell line THP-1-KO-EP B41-3 were as described in Seq-No.14 and Seq-No.15, respectively, and the genomic DNA from cells was extracted by ranh for PCR amplification.

(3) The PCR amplified product (about 200bp) was detected by 1% agarose gel electrophoresis (see FIG. 3), and the band of interest was excised.

(4) PCR amplification product Gel was recovered (FastPure Gel DNA Extraction Mini Kit cat # DC 301-01).

(5) The gel recovery product was ligated to a plasmid (pClone007 Versatile Simple Vector Kit cat # 007 VS).

(6) And (3) transformation: the rapid DH5 alpha competent cells were thawed on ice, the plasmid of the ligated product was transformed into competent cells, plated, monoclonal colonies were picked and sent to the department of science and technology for sequencing, 6 monoclonals were picked for each cell line. (results are shown in FIG. 4)

As can be seen from fig. 4: after the targeting effect of gRNA, the genome DNA lacks the base A, so that the frame shift mutation of the genome DNA is caused, and the protein 4.1R cannot be normally synthesized.

Example 3 Western Blot to detect expression of 4.1R +/+ THP-1 and 4.1R-/-T HP-1 cells at 4.1R protein level

(1) Extraction of Total cellular protein

Collecting THP-1 cells with good logarithmic phase state, washing twice with 1 × PBS, centrifuging at 800rpm for 5min, and discarding the supernatant; according to Western lysate: the ratio of protein inhibitors (PMSF) is 100: 1 preparing cell lysate, adding the cell lysate into collected cells, uniformly blowing and beating the cells, and cracking the cells on ice for 30min and shaking the cells once every 5min to fully crack the cells; centrifuging at 12000rpm for 10min at 4 deg.C, sucking supernatant into 1.5mL centrifuge tube, collecting a small part of protein for detecting protein concentration, adding 5 xSDS Loading Buffer into the rest, boiling in boiling water for 5min, subpackaging and storing at-80 deg.C in refrigerator for use.

(2) Protein concentration determination

The protein concentration determination is carried out according to the BCA protein concentration determination kit, and the specific steps are as follows:

preparing a working solution: according to the number of the standard and the sample, according to the reagent A: reagent B ═ 50: preparing a BCA working solution according to the proportion of 1, and fully and uniformly mixing; diluting the standard substance: mu.L of BSA standard was diluted to 100. mu.L with 1 XPBS to a final concentration of 0.5 mg/mL. Adding the standard substance into a 96-well plate according to 0, 2, 4, 6, 8, 12, 16 and 20 mu L per well, and adding 1 XPBS to make up to 20 mu L; diluting the samples, adding 20 μ L of sample per well to a 96-well plate, making 3 replicate wells per sample; sequentially adding 200 mu L of BCA working solution into each hole, incubating for 1h in an incubator at 37 ℃, reading the OD value at 570nm by using an enzyme-labeling instrument, and recording the reading; and (5) making a standard curve, and calculating the protein concentration of the sample to be detected according to the standard curve.

(3)Western Blot

Firstly, glue making

The 10% separation gel was formulated according to the instructions.

Carefully adding the separation glue into a glass plate to avoid bubbles, remaining 2mL of upper space, sealing with ultrapure water, standing at room temperature for 30min, and when a refraction line exists between the water and the glue, indicating that the glue is solidified. The ultrapure water was poured off, and the water was removed by blotting with absorbent paper.

Then the concentrated glue is prepared according to the instruction.

Adding the prepared concentrated glue, inserting into a comb to avoid generating bubbles, and standing at room temperature for 30 min; after the upper layer is solidified, the comb teeth are pulled out for electrophoresis.

② sample loading and electrophoresis

And (3) putting the rubber plate into an electrophoresis tank, adding 1 Xelectrophoresis buffer solution, slowly pulling out a comb, and adding a protein Marker and a protein sample. Starting electrophoresis at a constant voltage of 80V, changing the voltage to 120V when the bromophenol blue reaches the separation gel, and ending the electrophoresis when the bromophenol blue reaches the 1cm position at the lowest end of the separation gel;

③ half-dry rotor

And after electrophoresis is finished, opening the gel plate, taking out the protein gel, cutting the protein gel with corresponding size at the position corresponding to the target protein according to the indication of a protein marker, shearing the PVDF membrane into a shape consistent with the size of the protein gel, soaking in methanol for 1-2min, and then soaking in a membrane conversion buffer solution for 20min together with the protein gel. Sequentially putting the materials into an electric rotary tank: filter paper → PVDF membrane → protein glue → filter paper, compress tightly in the film transfer groove, after the positive and negative correspond well, transfer the film for 15 min;

is sealed

Preparing 5% of skimmed milk powder by using 1 × TBST, taking out the PVDF membrane after the membrane conversion is finished, marking, and placing on a shaking table to seal for 2 hours at room temperature;

fifthly, incubation primary antibody

Mixing the raw materials in a ratio of 1: dilution of 4.1R antibody with 5% skim milk powder at 1000 ratio, 1: the GAPDH antibody is diluted by 1000 proportion, the sealed PVDF membrane is placed in an incubation box, corresponding antibody diluent is added, and the incubation is carried out in a refrigerator at 4 ℃ overnight. Washing the membrane with 1 × TBST for 5min every time for 4 times;

sixth, incubation second antibody

Using 5% skimmed milk powder according to the weight ratio of 1: diluting a secondary antibody goat anti-rabbit IgG-HRP at a ratio of 4000, putting the PVDF membrane into the diluted secondary antibody, incubating at room temperature for 1h, and washing with 1 × TBST for 4 times after incubation, wherein each time lasts for 5-10 min;

seventhly, ECL luminescence development

After the membrane washing is finished, the ECL luminescent liquid is mixed according to the ratio of the liquid A: solution B is 1: 1, uniformly mixing, uniformly dripping the mixed luminescent liquid on a PVDF membrane, and exposing by using a full-automatic gel imager to obtain a protein band picture (the result is shown in figure 5). EPB41 knockout cell line and THP-1 cell line transfected with lentiCRISPR V2 plasmid not ligated to gRNA (as a positive control for subsequent experiments), in which lanes V2 are THP-1 cell lines transfected with lentiCRISPR V2 plasmid not ligated to gRNA, and lanes g1, g2 and g3 are THP-1-KO-EPB41-1, THP-1-KO-EPB41-2 and THP-1-KO-EPB41-3 cell lines, respectively.

Example 4 Effect of protein 4.1R on HIV infection

1. 4.1R +/+ THP-1(V2) and 4.1R-/-THP-1(g1) cells were plated at 3X 105/well in 12-well plates.

2. After the virus HIV MOI 1 was added to each well and adsorbed at 37 ℃ for 6 hours in a 5% CO2 incubator, the cell suspension was collected in a 1.5mL centrifuge tube, centrifuged at 800rpm for 5min, and the supernatant was discarded. Wash 2 times with 1 × PBS and once with serum-free medium.

3.10% FBS RPMI-1640 medium was resuspended, plated in corresponding 12-well plates, and incubated at 37 ℃ in a 5% CO2 incubator for 48 h.

4. After incubation for 24h at 37 ℃ in a 5% CO2 incubator for 48h, the cell suspension was collected in a 1.5mL centrifuge tube, centrifuged at 800rpm for 5min, and the supernatant was discarded. Intracellular RNA was extracted by Trizol method after 3 washes with 1 XPBS.

5. Real-time fluorescent quantitative PCR detection of viral load of HIV in V2 and g1 cells:

the fluorescent quantitative PCR reaction preparation system comprises the following steps:

setting a program: pre-denaturation at 90 ℃ for 30 s; at 61 deg.C for 20 min; at 95 ℃ for 30 s; 95 ℃ for 15 s; 60 ℃ for 1 min; 45 cycles.

The experimental results are shown in fig. 6:

after HIV infects V2 and g1 cells for 24h and 48h, the virus load in g1 cells is higher than that of V2 cells, which shows that 4.1R deletion can affect the HIV infection THP-1 cells, therefore, 4.1R-/-THP-1 established by the method can be used for researching the function of protein 4.1R on virus infection and antiviral immune response.

Sequence listing

<110> Zhengzhou university

KUNMING INSTITUTE OF ZOOLOGY, CHINESE ACADEMY OF SCIENCES

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Claims

1. A method for constructing an EPB41 gene knockout THP-1 cell line based on a CRISPR-Cas9 system is characterized by comprising the following steps: the method takes the protein coding sequence in the 1 st exon, the protein coding sequence in the 4 th exon and the protein coding sequence in the 7 th exon of the human EPB41 gene as the target sequence, and takes the ' N18 or N20 ' of the fragment which accords with the sequence rule arrangement of 5 ' -G-N18-NGG-3 or 5 ' -G-N20-NGG-3 ' or 5 ' -CCN-N18-C-3 ' or 5 ' -CCN-N20-C-3 ' as the target sequence; n represents any one of A, T, C, G, where "N18" and "N20" are 18 and 20 deoxynucleotides, respectively.

2. The method for constructing the EPB41 gene knockout THP-1 cell line based on the CRISPR-Cas9 system according to claim 1, wherein the method comprises the following steps: the target sequences are shown as Seq-No.1, Seq-No.2 and Seq-No. 3.

3. The method for constructing the EPB41 gene knockout THP-1 cell line based on the CRISPR-Cas9 system according to claim 2, wherein the method comprises the following steps: the target sequence Seq-No.1 is positioned on a first exon sequence of the EPB41 gene; the sequence of Seq-No.2 is located in the 4 th exon sequence of EPB41 gene; the sequence of Seq-No.3 is located in the 7 th exon sequence of the EPB41 gene.

4. The method for constructing the EPB41 gene knockout THP-1 cell line based on the CRISPR-Cas9 system according to claim 3, wherein the method comprises the following steps: the method is the following method A or method B or method C:

method A (target sequence SEQ ID No.1) comprising the steps of:

(a1) design of sgRNA and synthesis of oligonucleotide chain, and synthesis of oligonucleotide chain 1 sgRNA is designed according to the first exon of human EPB41 gene, and 2 single-stranded DNAs named as forward single-stranded DNA-1 and reverse single-stranded DNA-1 are synthesized, wherein the sequence of the forward single-stranded DNA-1 is shown as Seq-No. 4; the sequence of the forward single-stranded DNA-1 is as defined in Seq-No. 5;

method B (target sequence SEQ ID No.2) comprising the steps of:

(b1) designing sgRNA and synthesizing oligonucleotide chains, and synthesizing 1 sgRNA according to the fourth exon of the human EPB41 gene to synthesize 2 single-stranded DNAs named as forward single-stranded DNA-2 and reverse single-stranded DNA-2, wherein the forward single-stranded DNA-2 has a sequence shown as Seq-No. 6; the sequence of the forward single-stranded DNA-2 is as defined in Seq-No. 7;

(b2) carrying out annealing reaction on the forward single-stranded DNA-2 and the reverse single-stranded DNA-2 to obtain double-stranded DNA-2;

method C (target sequence SEQ ID No.3), comprising the steps of:

(c1) design of sgRNA and synthesis of oligonucleotide chain, and synthesis of oligonucleotide chain 1 sgRNA is designed according to the seventh exon of human EPB41 gene, and 2 single-stranded DNAs named as forward single-stranded DNA-3 and reverse single-stranded DNA-3 are synthesized, wherein the sequence of the forward single-stranded DNA-3 is shown as Seq-No. 8; the sequence of the forward single-stranded DNA-3 is as defined in Seq-No. 9;

(c2) carrying out annealing reaction on the forward single-stranded DNA-3 and the reverse single-stranded DNA-3 to obtain double-stranded DNA-3;

5. The method for constructing the EPB41 gene knockout THP-1 cell line based on the CRISPR-Cas9 system according to claim 4, wherein the method comprises the following steps: in the step (a3), the step (B3) and the step (c3), the plasmids are LentiCRISPR V2(B) -gRNA-EPB41-1, LentiCRISPR V2(B) -gRNA-EPB41-2 and LentiCRISPR V2(B) -gRNA-EPB41-3, respectively.

6. EPB41 gene-knocked-out THP-1 cell line THP-1-KO-EPB41-1, THP-1-KO-EPB41-2 and THP-1-KO-EPB41-3 established by the method for constructing the EPB41 gene-knocked-out cell line based on the CRISPR-Cas9 system as claimed in any one of claims 4 and 5, and applications thereof.