CN110964744A

CN110964744A - Human osteosarcoma U-2OS tool cell line capable of stably expressing Cas9 protein and preparation method and application thereof

Info

Publication number: CN110964744A
Application number: CN201911336354.2A
Authority: CN
Inventors: 蒋明贵; 周勇华; 黄文涛; 刘军鹏; 蔡艳; 刘彩云; 许澎; 屈飞
Original assignee: Hunan Pratzer Biotechnology Co Ltd
Current assignee: Hunan Pratzer Biotechnology Co Ltd
Priority date: 2019-12-23
Filing date: 2019-12-23
Publication date: 2020-04-07

Abstract

The invention discloses a human osteosarcoma U-2OS tool cell strain capable of stably expressing Cas9 protein, a preparation method and application thereof, wherein the preparation method comprises the steps of introducing a Cas9 protein expression gene and an antibiotic screening marker gene into a CRISPR/Cas9 system to construct a human osteosarcoma U-2OS tool cell strain capable of stably expressing Cas9 protein; the cell strain can stably express Cas9 protein, relevant gene editing work can be carried out only by transfecting single sgRNA, only a small sgRNA fragment needs to be transfected when specific gene editing is carried out, and the efficiency of gene editing can be greatly improved. Osteosarcoma is the most common primary malignant bone tumor, U-2OS is a common osteosarcoma cell model, and the gene engineering cell strain provided by the invention has important significance for researching pathogenesis and regulation network of osteosarcoma and preventing and early treating bone tumor.

Description

Human osteosarcoma U-2OS tool cell line capable of stably expressing Cas9 protein and preparation method and application thereof

Technical Field

The invention belongs to the field of genetic engineering and genetic modification, and particularly relates to a human osteosarcoma U-2OS tool cell strain capable of stably expressing Cas9 protein, and a preparation method and application thereof.

Background

Osteosarcoma is the most common primary malignant bone tumor in a skeletal system originated from mesenchymal tissues, and has a high incidence rate in adolescents and aged over 60 years, which can reach 35% of primary bone tumor. Moreover, osteosarcoma has the characteristics of high malignancy, early metastasis and poor prognosis. Currently, the treatment of osteosarcoma is mainly based on surgery and chemotherapy. With the development of neoadjuvant chemotherapy, the overall survival rate of osteosarcoma patients has currently increased from 10% to 70%. However, there are still around 30% of patients who experience relapse and metastasis, resulting in treatment failure. U-2OS was originally isolated from a sarcoma differentiated from the tibia of a 15 year old girl in 1964 and developed into a tool cell line commonly used in osteosarcoma cell models. The U-2OS cell is used as a model to research the pathogenesis and the regulation network of osteosarcoma, and has important significance for prevention and early treatment.

The CRISPR system is an immune system against exogenous genetic material owned by prokaryotes. By having specific sequences, RNA-mediated targeting of specific targets, CRISPR systems are capable of cleaving exogenous DNA, including phages and exogenous plasmids, resulting in the loss or partial loss of function of the gene of interest. The CRISPR/Cas system can be used as a site-directed gene editing system, has the characteristics of simple operation, low cost and high effect, and is a new gene editing technology which is widely used for basic research at present. The CRISPR/Cas system is a powerful tool for gene editing, allowing precise editing of the genomes of most species. When a plasmid containing a Cas9 protein encoding gene and a sgRNA expression plasmid are co-transfected into a certain cell, the sgRNA can target a target sequence, and the Cas9 protein can break a corresponding DNA double strand. The organism itself has a response mechanism of DNA damage repair, and will link the sequences at the upstream and downstream ends of the break. Since base insertion or deletion is likely to occur during repair, the repaired base sequence is often different from the original base sequence, and thus the target gene in the cell is changed. If a repair template plasmid (donor DNA molecule, i.e.the donor vector) is introduced into the cell on this basis, the cell will, by homologous recombination, introduce fragments or site-directed mutations in the repair process according to the supplied template, and thus achieve gene replacement or mutation.

However, since the Cas9 protein coding gene is large and close to 4000bp, the transfection efficiency is often low due to large plasmid during gene editing, the gene editing efficiency is seriously affected, and the success rate of constructing a gene editing cell line/gene editing mode animal is greatly reduced. Therefore, it is very important to construct a new gene editing system.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention provides a preparation method of a human osteosarcoma U-2OS tool cell strain capable of stably expressing Cas9 protein, which constructs a genetic engineering cell strain capable of stably expressing Cas9 protein and carrying a puromycin resistance selection marker based on a CRISPR/Cas9 system.

The invention also provides a human osteosarcoma U-2OS tool cell strain prepared by the preparation method.

The invention also provides application of the human osteosarcoma U-2OS tool cell strain.

The preparation method according to the embodiment of the first aspect of the invention comprises the following steps:

s1, designing and constructing a fixed-point cutting vector capable of expressing the target sgRNA; the nucleotide sequence of the sgRNA is shown as SEQ ID No. 1;

s2, constructing a donor vector containing a Cas9 protein expression gene, an AAVS1 gene homologous sequence and an antibiotic selection marker gene;

s3, transferring the site-specific cutting vector and the donor vector into a human osteosarcoma U-2OS cell, screening positive clones through antibiotic resistance, and identifying to obtain the gene.

The preparation method provided by the embodiment of the first aspect of the invention has at least the following beneficial effects: the invention relates to a human osteosarcoma tool cell U-2OS gene engineering cell strain which is constructed by a CRISPR/Cas9 system and can stably express Cas9 protein; the donor vector with a specific fragment (AAVS1 site) is used for accurately integrating the exogenous sequence into a specific site, so that the risk caused by random integration of viral elements can be effectively avoided; the human osteosarcoma cell strain prepared by the method can stably express Cas9 protein, has scientific research significance and market value for quickly carrying out gene editing of target genes, researching the influence of the target genes on the pathogenesis of osteosarcoma and searching treatment targets.

According to some embodiments of the invention, the antibiotic selection marker gene is a puromycin resistance gene.

According to some embodiments of the invention, the AAVS1 gene homology sequences include HA-L and HA-R; the nucleotide sequence of the HA-L is shown as SEQ ID NO. 2; the nucleotide sequence of the HA-R is shown in SEQ ID NO. 3.

Preferably, the donor vector further comprises elements T2A, FLAG, NLS and poly (a); the connection sequence of the elements is HA-L-T2A-FLAG-NLS-Cas9-NLS-T2A-puro R-Poly (A) -HA-R. The Cas9 protein expression gene is connected with HA-L of AAVS1 through T2A; cas9 protein expression gene and puromycin resistance gene puro R are connected through an element T2A, and a poly (A) structure is carried behind the puro R and can automatically terminate the expression of the inserted element. Cas9 protein carries a nuclear entry signal NLS, and can enter the nucleus to play a biological function.

Preferably, the structure of the donor vector is HA-L-T2A-3X FLAG-SV40 NLS-Cas9-NLS-T2A-puro R-Poly (A) -HA-R.

According to some embodiments of the invention, the nucleotide sequence of the Cas9 protein expression gene is shown as SEQ ID No. 4.

According to some embodiments of the present invention, the site-specific excision vector comprises a fluorescent marker gene, wherein the fluorescent marker gene is a GFP fluorescent marker gene; preferably, the method further comprises performing a culturing operation (preferably culturing for 2 weeks) after the screening is completed, and performing flow sorting on the cultured cells to sort out GFP-negative cells.

The human osteosarcoma U-2OS tool cell line according to the second aspect of the present invention is prepared by the above-mentioned preparation method.

The human osteosarcoma U-2OS tool cell line according to the embodiment of the second aspect of the present invention has at least the following beneficial effects: the human osteosarcoma U-2OS tool cell strain contains a Cas9 protein expression gene, can solve the problem of low transfection efficiency caused by large plasmid during gene editing due to large Cas9 protein coding gene, and improves the gene editing efficiency; osteosarcoma is the most common primary malignant bone tumor, and the pathogenesis and the regulation and control network of osteosarcoma can be further researched by using U-2OS cells as a model, so that the osteosarcoma has important significance for preventing and treating bone tumor as soon as possible.

According to a third aspect of the present invention there is provided a method of preparing a gene-editing cell line, comprising the steps of: and (3) carrying out subsequent gene editing operation by using the human osteosarcoma U-2OS tool cell line as a tool cell to obtain the gene editing cell line.

The application of the embodiment of the third aspect of the invention has at least the following beneficial effects: the cell strain for gene editing prepared by using the human osteosarcoma U-2OS tool cell strain has simple steps, and can express Cas9 protein, so that only a corresponding sgRNA fragment needs to be transferred, the influence of the target gene on the pathogenesis of osteosarcoma can be researched, and the research on the effect of the target gene on the pathogenesis of osteosarcoma can be realized quickly, and the research on the therapeutic target point has scientific research significance and market value.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

FIG. 1 is a map of a site-directed cleavage vector (sgRNA vector) in example 1 of the present invention.

FIG. 2 is a map of a donor vector constructed in example 1 of the present invention.

FIG. 3 is a schematic view of the functional elements of the donor vector in example 1 of the present invention.

FIG. 4 is a graph showing the effect of U-2OS electrotransformation in example 1 of the present invention (both the sgRNA vector transfected and the Donor vector fluoresce green).

FIG. 5 is a diagram of flow-sorted GFP negative U-2OS single cells in example 1 of the present invention.

FIG. 6 is a diagram of a monoclonal cell line formed from single cells that were flow sorted in example 1 of the present invention.

FIG. 7 is a diagram showing the genome identification results of the human osteosarcoma cell line U-2OS genetically engineered cell strain with Cas9 protein and antibiotic selection marker (puromycin) in example 1 of the present invention.

Detailed Description

In order to explain technical contents, achieved objects, and effects of the present invention in detail, the following description is made with reference to the accompanying drawings in combination with the embodiments.

Materials and biochemical reagents used in examples are all conventional commercially available reagents unless otherwise specified, and technical means used in examples are well known to those skilled in the art.

Example (b): construction of human osteosarcoma U-2OS tool cell strain capable of stably expressing Cas9 protein

Firstly, designing and constructing a site-specific cleavage vector capable of expressing target sgRNA

1.1 extraction of cleavage vector Primary plasmid

Site-directed cleavage vector 1 original plasmid (purchased from addrene, cat #71814) was extracted in small quantities using a plasmid miniprep kit (purchased from seikang as a century, cat # CW0511C) following the experimental procedures of the product instructions.

1.2 cleavage of vector original plasmid by digestion

The plasmid extracted at 1.1 was digested with an endonuclease (purchased from Neb, cat # R0539L) according to the experimental procedures of the product instructions, and the digested product was recovered.

1.3 construction of sgRNA target sequence fragments

sgRNA target sequence primers were synthesized and annealing was performed by a PCR instrument.

1.3.1 primers for the designed sgRNA target sequence were as follows:

sg-F：5`-3`:CACCGTCACCAATCCTGTCCCTAG；

sg-R：5`-3`:AAACCTAGGGACAGGATTGGTGAC。

1.3.2 annealing reactions use the procedure:

a.95℃，5min；

b.25℃，20min。

1.3.3 the annealed product was diluted to 10. mu.M for subsequent experiments.

1.4 construction of cleavage vectors

1.4.1 ligation of the digested original plasmid and diluted sgRNA target sequence was performed using T4 ligase (purchased from Neb under stock No. M0202S) according to the product instructions.

1.4.2 transformation experiments were performed on the ligation-completed mixtures using competent cells (purchased from Bomaide, cat # BC102-02) according to the product instructions.

1.4.3 picking single clone to sequence, comparing the sequencing result, selecting the successfully constructed site-directed cutting vector, the map is shown in figure 1.

Second, construction of Donor vector

A donor vector containing Cas9 protein and an antibiotic selection marker (puromycin) was constructed.

2.1 extraction of Donor (donor) vector original plasmid

Donor (donor) vector original plasmid PAAVS1-P-CAG-GFP (purchased from Addge, cat #80491 and pSpCas9(BB) -2A-Puro (PX459) V2.0 (purchased from Addge, cat #124286) was extracted in small amounts using a plasmid miniprep kit (purchased from Kanghong as a century, cat # CW0511C) following the experimental procedures of the product instructions.

2.2PCR amplification of fragments of interest

2.2.1 amplification of the recombinant armed 3Xflag-cas9-T2A-puroR fragment from the pSpCas9(BB) -2A-Puro (PX459) V2.0 vector.

The amplification primers were designed as follows:

Cas9-F1：ATCCCGGCCCTAGGCTCGAGGACTATAAGGACCACGACGG；

Cas9-R1：TGGCAGAGGGAAAAAGATCTAGCGAGCTCTAGTTAGAATTCTCAG；

the PCR product size was 4996 bp.

2.2.2 amplification vector PAAVS1-P-CAG-GFP backbone fragment.

The amplification primers were designed as follows:

Cas9-F2:AGATCTTTTTCCCTCTGCCAAAAATTATGG；

Cas9-R2:CTCGAGCCTAGGGCCGGGATTCTCC；

the PCR product size was 6216 bp.

2.2.3 recombinant ligation

2.2.3.1 the two fragments obtained in 2.2.2 (4996bp and 6216 bp) were recombinantly ligated using a seamless ligase (purchased from Ogaku corporation, cat # TSV-S2) according to the procedures of the product instructions;

2.2.3.2 transformation experiments were performed on the ligation-completed mixtures using competent cells (purchased from Bomaide, cat # BC102-02) following the procedures of the product instructions.

2.2.3.3 selecting a single clone for sequencing, comparing sequencing results, selecting and constructing a successful donor vector, wherein a vector map is shown in figure 2, a Cas9 protein expression gene and an antibiotic resistance gene in the donor vector are connected through an element T2A, and the Cas9 protein expression gene also comprises a nuclear localization signal sequence NLS, and can enter a cell nucleus to play a biological function.

Wherein, the donor vector contains AAVS1 gene homologous sequence, including homologous left arm sequence (HA-L) and homologous right arm sequence (HA-R), the nucleotide sequence of the homologous left arm is shown in SEQ ID NO.2, and the nucleotide sequence of the homologous right arm is shown in SEQ ID NO. 3; the Cas9 protein expression gene comprises an independent constitutive promoter and a termination structure, and the nucleotide sequence of the Cas9 protein expression gene is shown as SEQ ID No. 4.

The AAVS1 locus is located on chromosome 19 of the human genome and studies have shown that: the gene transferred by the site can be normally and stably expressed, and is the most frequently used site for site-specific integration in human cells. The CRISPR/Cas9 system specifically targeting the AAVS1 site can generate DNA double-strand breaks at the AAVS1 site on the human chromosome 19, trigger the DNA repair mechanism of cells, induce Homologous Recombination (HR) between a genome and an AAVS1 donor plasmid, and integrate a DNA fragment on the donor plasmid into the AAVS1 site on the genome.

Thirdly, transferring the site-specific excision vector and the donor vector into a tool cell U-2OS cell

3.1 culturing U-2OS cells

The U-2OS cells were recovered and cultured. Cell culture media are shown in the following table.

TABLE 1 complete Medium composition of U-2OS cells

The U-2OS cells used in this example were identified by STR and determined to be authentic U-20S cells.

3.2 transfection of cells

The embodiment adopts an electrotransformation instrument H1 of Jiangsu Yida biology to carry out electrotransformation experiments according to the experimental parameters and the operation instructions recommended by manufacturers.

3.2.1 before electrotransfer 30min, Buffer, cell culture solution and D-PBS were taken out in advance and returned to room temperature.

3.2.2 cell harvest, count: uniformly suspending U-20S cells in a logarithmic growth phase, placing the cells in an EP tube, and counting by trypan blue staining;

3.2.3 centrifugation: take 2x10⁶Placing the cells in a new centrifugal tube, centrifuging at 1000rpm for 5 min;

3.2.4D-PBS Wash: removing the supernatant culture solution to obtain the required cells, adding 1mL of D-PBS to resuspend the cells, centrifuging at 1000rpm for 5 min;

3.2.5DNA, cells, buffer mix: discarding the D-PBS, adding required amount of buffer and plasmid, and gently blowing, beating and uniformly mixing;

3.2.6 electrotransfer: adding the cell suspension mixed with the plasmid into an electric rotating cup, covering the electric rotating cup with a cover according to 100 mul/hole, inserting the electric rotating cup into an electrode base groove, and performing electric rotation under a proper electric rotation condition;

the electrotransfer conditions were: the voltage is 250V, 6 pulses, the pulse time is 800ms, and the time interval is 1000 ms.

3.2.7 detection: cell fluorescence expression was observed under a fluorescence microscope 48 hours after the electrotransfer. In a microscope visual field, the more cells with positive green fluorescence proportion, the stronger green fluorescence, which indicates the higher electrotransfer efficiency (the fixed point cutting vector is provided with a GFP label), the electrotransfer effect is shown in FIG. 4, and the stronger green fluorescence indicates the high electrotransfer efficiency of the operation process.

3.3 puromycin screening Positive clones

Cells transfected with 3.2 cells were used and selected by adding puromycin to the complete medium of normal U-2OS cells (see Table 1). The puromycin screening concentration used in the invention is 2 mug/ml, and the screening time is 72 hours.

After the selection was completed, the medium was changed to normal U-2OS cell complete medium for 2 weeks. Through puromycin screening, cells which are transfected with the site-specific excision vector and the donor vector successfully can be enriched.

3.4 flow sorting

The cells obtained after screening in 3.3 were single cell sorted using a flow cytometer to ensure that each well contained only 1 cell as shown in figure 5. Through flow sorting, single cells which are integrated with Cas9 protein expression genes and antibiotic selection markers in a fixed point mode can be further purified and obtained.

3.5 Single cell construction line

3.5.1 Single cells obtained in 3.4 were cultured using normal U-2OS cell complete medium until a single cell-derived monoclonal cell subline was grown, as shown in FIG. 6 and designated U-2OS-cas 9.

3.5.2 picking 5 cell sublines from single cell source for expanding culture, and respectively naming the sublines as U-2OS-Cas9-1, U-2OS-Cas9-2, U-2OS-Cas9-3, U-2OS-Cas9-4 and U-2OS-Cas 9-5.

3.6 identifying and obtaining the human osteosarcoma cell U-2OS genetic engineering cell strain which is constructed based on the CRISPR/Cas9 system and stably expresses the Cas9 protein through a molecular biology experiment.

3.6.1 genomic PCR identification of Positive clones

a. 5 subcloned cell lines of 3.5.2 were collected and the genomes were extracted separately using a genome extraction kit (purchased from Tiangen, cat # DP 304-03).

b. Designing a genome identification primer, and carrying out PCR amplification on the extracted genome.

genome-F1: GCATTGGAGTCGCTTTAACTG;

genome-R1: GGTTCTTCTTGATGCTGTGCC;

genome-R2: CACAGGTAAAACTGACGCACG.

Next, the amplified DNA product was identified by gel electrophoresis, and the identification chart is shown in FIG. 7.

As shown in the figure: subcloned cell lines 1 and 3(U-2OS-Cas9-1, U-2OS-Cas9-3) were identified as false positives with no integration of the relevant gene fragment in the genome; subcloned cell lines 2, 4, 5 were positive clones. (FIG. 7, left panel, using primer pair F1R1, 1545bp, R1 on cas9, amplified fragment genome sequence + partial vector sequence). And the subcloned cell line U-2OS-Cas9-4 is homozygous positive; the subcloned cell lines U-2OS-Cas9-2, U-2OS-Cas9-5 were heterozygous positive (shown on the right of FIG. 7, using primer pairs F1R2, 1653bp, R2 on HA-R, amplified fragments were normal genomic sequences). Therefore, the subcloned cell line U-2OS-Cas9-4 was chosen as a tool cell line for subsequent experiments.

Osteosarcoma is the most common primary malignant bone tumor, and U-2OS is a common cell model of osteosarcoma. By using the U-2OS cell as a model, the human osteosarcoma U-2OS tool cell line provided by the embodiment has important significance for researching pathogenesis and regulation network of osteosarcoma and preventing and early treating bone tumor. The cell strain can stably express Cas9 protein, related gene editing work can be carried out only by transfecting single sgRNA, only a small sgRNA fragment needs to be transfected when specific gene editing is carried out, and the gene editing efficiency can be greatly increased.

In conclusion, the human osteosarcoma U-2OS tool cell strain which is constructed based on the CRISPR/Cas9 system and can stably express the Cas9 protein has scientific research significance and market value for rapidly researching the influence of a target gene on the pathogenesis of osteosarcoma and searching a therapeutic target.

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent changes made by using the contents of the present specification and the drawings, or applied directly or indirectly to the related technical fields, are included in the scope of the present invention.

Sequence listing

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<120> human osteosarcoma U-2OS tool cell strain capable of stably expressing Cas9 protein and preparation method and application thereof

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gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac 2580

aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac 2640

tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc 2700

aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg 2760

gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact 2820

aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag 2880

ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac 2940

caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac 3000

cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg 3060

atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac 3120

atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct 3180

ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc 3240

accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag 3300

acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc 3360

agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat 3420

tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa 3480

gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt 3540

ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac 3600

tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag 3660

aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac 3720

tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag 3780

cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc 3840

ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc 3900

atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct 3960

gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag 4020

gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac 4080

ctgtctcagc tgggaggcga c 4101

Claims

1. A preparation method of a human osteosarcoma U-2OS tool cell line capable of stably expressing Cas9 protein is characterized by comprising the following steps:

s1, designing and constructing a fixed-point cutting vector capable of expressing the target sgRNA; the nucleotide sequence of the sgRNA is shown in SEQ ID NO. 1;

2. The method according to claim 1, wherein the antibiotic selection marker gene is a puromycin resistance gene.

3. The method of claim 1, wherein the AAVS1 gene homology sequences include HA-L and HA-R; the nucleotide sequence of the HA-L is shown as SEQ ID NO. 2; the nucleotide sequence of the HA-R is shown in SEQ ID NO. 3.

4. The method of claim 3, wherein said donor vector further comprises elements T2A, FLAG, NLS, and Poly (A); the connection sequence of the elements is HA-L-T2A-FLAG-NLS-Cas9-NLS-T2A-puro R-Poly (A) -HA-R; puro R is puromycin resistance gene.

5. The preparation method of claim 1, wherein the nucleotide sequence of the Cas9 protein expression gene is shown as SEQ ID No. 4.

6. The method according to claim 1, wherein the site-specific cleavage vector comprises a fluorescent marker gene, and the fluorescent marker gene is a GFP fluorescent marker gene.

7. The method of claim 6, further comprising culturing the cells after the screening step, and performing flow sorting of the cultured cells to sort out GFP negative cells.

8. Human osteosarcoma U-2OS tool cell line produced by the production method according to any one of claims 1 to 7.

9. A method of preparing a gene-editing cell line comprising the steps of: using the human osteosarcoma U-2OS tool cell line of claim 8 as a tool cell, performing a subsequent gene editing operation to obtain the gene editing cell line.