WO2020000437A1

WO2020000437A1 - Method for knocking out human aprf gene

Info

Publication number: WO2020000437A1
Application number: PCT/CN2018/093841
Authority: WO
Inventors: 毛吉炎
Original assignee: 深圳市博奥康生物科技有限公司
Priority date: 2018-06-29
Filing date: 2018-06-29
Publication date: 2020-01-02

Abstract

Disclosed is a method for knocking out the human APRF gene by using CRISPR-Cas9 gene editing technology. The method comprises the following specific operation steps: (1) design of a sgRNA sequence, (2) ligation, transformation and amplification of the sgRNA, (3) plasmid transfection of 293T cells and packaging same into lentiviruses, (4) lentiviral infection of the target cell and screening same with puromycin, and (5) verification of the APRF gene knockout result.

Description

Method for knocking out human APRF gene

Technical field

The invention mainly relates to the field of genetic engineering, in particular to a method for knocking human APRF genes by using CRISPR-Cas9 gene editing technology.

Background technique

Signal transducers and transcriptional activators (STAT) are important DNA-binding proteins in cells. These proteins have dual roles and are transcription factors for cytoplasmic signal transductors and the nucleus. APRF is an important member of the STAT family and is closely related to cell proliferation, apoptosis and differentiation. Under normal conditions, the activation of APRF by extracellular signals is strictly regulated. The process of APRF activation is short and rapid, which can maintain the biological function of normal cells. Deletion of APRF causes death of early embryonic cells.

technical problem

In many tumors, such as leukemia, breast cancer, head and neck tumors, malignant melanoma, prostate cancer, and pancreatic cancer, APRF is continuously activated. Studies have shown that continued activation of APRF contributes to tumor formation. Continuous activation of APRF can mediate tumor immune escape, promote tumor neovascularization, inhibit tumor cell apoptosis, promote tumor cell proliferation and other mechanisms, leading to malignant transformation of tumor cells. Therefore, APRF is an ideal target for the treatment of potential tumors, but the lack of a method for specifically knocking out human APRF genes in the prior art has caused a certain obstacle to the progress of related research.

Technical solutions

In order to achieve the purpose of the present invention, the present invention provides a method for knocking out human APRF gene by using CRISPR-Cas9 gene editing technology. The specific operation steps are as follows:

(1) Design the sgRNA sequence

In the APRF gene expression DNA region, near the N-terminus of the protein coding region, find the sequence that starts with NGG, and take the upstream 20 The nt sequence is used as the sgRNA to be selected to ensure that it has no homology or low homology with the sequences of other genes, and its sequence is shown in SEQ ID NO.1. According to actual needs, two strands of sgRNA need to be synthesized separately for subsequent connection, and the sequences of the two strands are shown in SEQ ID NO. 2 and SEQ ID NO. 3, respectively. Entrust the company to synthesize these two sequences;

(2) Ligation, transformation and amplification of sgRNA

Dilute 2 synthetic single-stranded sgRNA sequences to After 100 μmol / L, dsDNA was mixed and annealed to form dsDNA, and then ligated to the lenti CRISPR v2 vector treated with BsmBI endonuclease.

The above products were transformed into E. coli competent cells Stbl3 according to the conventional molecular cloning technology method, and positive clones were selected. The positive clones were picked and expanded and cultured. A large number of plasmids were extracted to obtain a constructed CRISPR-Cas9 system containing the knockout APRF gene. Expression plasmid, save for later use;

(3) 293T cells transfected with plasmid and packaged into lentivirus

First thawed and cultured 293T cells. After passage of growth culture for 2 times, transfection was performed: CRISPR-Cas9 system expression plasmid containing APRF gene knockout and 1 μg each of the two helper plasmids were taken. Lipofectamine 3000 was co-transfected into 293T cells. 48 hours before transfection, inoculate cells into a well plate or petri dish for lentivirus production. During transfection, the confluence of cells is about 70% -80% is the best infection state, and the viability is ≥95%. The dyeing time was the starting point, and the harvest time was 48 h and 72 h. After filtering by μm filter, store at -80 ℃;

(4) Lentiviral infection and puromycin selection of target cells

Thaw culture HepG2 cells. When the confluence of the cells is about 70% -80%, add a mixture of lentivirus and culture medium (containing 4 μg / mL polybrene) to treat 24 After h, the lentiviral solution was changed to a complete medium containing 1 μg / mL puromycin, and the screening culture was started. 7-14 After d, the cells infected with lentivirus will form single cell clones, and the cell selection is completed.

(5) Validation of APRF gene knockout results

The selected HepG2 cells (experimental group) and normal HepG2 cells without any treatment were extracted respectively, and their genomic DNA was extracted and used as a template for PCR amplification. After re-annealing, they were treated with T7E1 enzyme and agarose gel electrophoresis Observe the results of APRF gene knockout.

Beneficial effect

The method for knocking out the APRF gene provided by the present invention and the cell strain constructed by applying the method provide an experimental technology platform for further exploring the role of the APRF gene, and can be used in research and development of drugs related to abnormal expression of APRF.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of the results of identifying the editing status of the APRF gene by the T7E1 enzyme.

Embodiments of the invention

The present invention is further described below with reference to specific examples, the examples are merely explanatory and are not meant to limit the scope of the present invention in any way.

实施例一：Embodiment one: sgRNAsgRNA 的设计the design of

In the APRF gene expression DNA region, near the N-terminus of the protein coding region, find the sequence that starts with NGG, and take the upstream 20 The nt sequence is used as the sgRNA to be selected to ensure that it has no homology or low homology with the sequences of other genes. Its sequence is 5’- GAAGGGCCTCTCCGAGCCGA -3 ’, such as SEQ ID NO.1. According to the actual needs, the two strands of sgRNA need to be synthesized separately: the CACC sequence needs to be added to the 5 'end of the sgRNA sense strand, and the AAAC sequence needs to be added to the 5' end of the sgRNA antisense strand for subsequent connection. The sequences of the two strands are 5 ' -CACCGAAGGGCCTCTCCGAGCCGA -3 'and 5'- AAACTCGGCTCGGAGAGGCCCTTC -3', such as SEQ ID NO. 2 and SEQ ID NO. 3 are shown. The company was commissioned to synthesize the two sequences.

实施例二Example two ：基因敲除载体的构建: Construction of gene knockout vectors

The synthesized two single-stranded sgRNA sequences were diluted to 100 μmol / L, mixed in equal amounts and annealed to form dsDNA, and then ligated to the lenti CRISPR v2 vector treated with BsmBI endonuclease.

The above products were transformed into E. coli competent cells Stbl3 according to the conventional molecular cloning technology method, and positive clones were selected. The positive clones were picked up and cultured, and then verified by sequencing to screen out positive clones E. coli containing sequences that fully matched the expected. It performs expansion culture, and then uses the endotoxin-free plasmid extraction kit to extract the recombinant vector therein, and a large number of constructed CRISPR-Cas9 system-containing expression vectors pLentiCRISPR-APRF are obtained.

实施例三：慢病毒的包装Example 3: Packaging of lentivirus

Thaw and culture 293T cells and wait for 2 passages of growth and culture, then perform transfection: take pLenti CRISPR-APRF plasmid and two lentiviral packaging helper plasmids each 1 μg, and use Lipofectamine 3000 was co-transfected into 293T cells. 48 hours before transfection, inoculate cells into a well plate or petri dish for lentivirus production. During transfection, the confluence of cells is about 70% -80% is the best infection state, and the viability is ≥95%. The dyeing time was the starting point, and the harvest time was 48 h and 72 h. After filtration with a μm filter, it was stored at -80 ° C.

实施例四：Embodiment 4: HepG2HepG2 细胞的慢病毒感染及嘌呤霉素筛选Lentiviral infection of cells and puromycin selection

Culture HepG2 cells. When the confluence of the cells is about 70% -80%, add a mixture of lentivirus and culture medium (containing 4 μg / mL polybrene) and treat 24 After h, the lentivirus solution was changed to a complete medium containing 1 μg / mL puromycin, and the screening culture was started. The screening time was 7-14. d. Change the fluid every other day. Cells infected with lentivirus will form single-cell clones, and the cell selection is complete.

实施例五：Embodiment 5: T7E1T7E1 酶鉴定Enzyme identification APRFAPRF 基因敲除结果Knockout results

The lentivirus-infected HepG2 cells (experimental group) and normal HepG2 cells (control group) were expanded and cultured. Genomic DNA was extracted and amplified by high-fidelity PCR. The PCR product was recovered by electrophoresis, and then the product was digested with T7 endonuclease I at 37 ° C for 1 h. After the digestion, 1% agarose gel electrophoresis was performed, and the results are shown in FIG. 1. It can be seen that the PCR product of the control group was still only one band after digestion, while the experimental group showed multiple bands, indicating that the APRF gene in HepG2 cells was successfully edited.

Industrial applicability

Claims

A method for knocking out human APRF genes, characterized in that the method includes the following operation steps:

(1) Design the sgRNA sequence

In the part of the APRF gene expression DNA that is near the N-terminus of the protein coding region, find the sequence that starts with NGG, and take the upstream 20 nt sequence as the candidate sgRNA to ensure that it has no homology or homology with the sequences of other genes The sex is very low, and its sequence is shown in SEQ ID NO.1. According to actual needs, two strands of sgRNA need to be synthesized separately for subsequent connection, and the sequences of the two strands are shown in SEQ ID NO. 2 and SEQ ID NO. 3, respectively. The company was commissioned to synthesize the two sequences.

(2) Ligation, transformation and amplification of sgRNA

After diluting the synthesized two single-stranded sgRNA sequences to 100 μmol / L, they were mixed and annealed to form dsDNA, and then ligated to the lenti CRISPR v2 vector treated with BsmBI endonuclease.

The above products were transformed into E. coli competent cells Stbl3 according to the conventional molecular cloning technology method, and positive clones were selected. The positive clones were picked and expanded and cultured. A large number of plasmids were extracted to obtain a constructed CRISPR-Cas9 system containing the knockout APRF gene. Expression plasmid, save for later use;

(3) 293T cells transfected with plasmid and packaged into lentivirus

First thawed and cultured 293T cells, and then transfected them after two passages of growth and culture: Take the expression plasmid of CRISPR-Cas9 system containing the knockout APRF gene and 1 μg each of the two helper plasmids, and co-transfect into 293T with Lipofectamine 3000 Cell. 48 hours before transfection, inoculate cells into a well plate or petri dish for lentivirus production. During transfection, the confluence of cells is about 70% -80% is the best infection state, and the viability is ≥95%. The staining time is the starting point. The harvest time is 48 h and 72 h, and the supernatant is harvested. After filtering through a 0.45 μm filter, it is stored at -80 ° C.

(4) Lentiviral infection and puromycin selection of target cells

Thaw culture HepG2 cells, pass through the growth culture twice, and when the confluence of the cells is about 70% -80%, add a mixture of lentivirus and culture medium (containing 4 μg / mL polybrene) for 24 h, and then lentivirus solution The medium was changed to a complete medium containing 1 μg / mL puromycin, and the screening culture was started. After 7-14 days, the cells infected with lentivirus will form single cell clones, and the cell selection is completed.

(5) Validation of APRF gene knockout results

The selected HepG2 cells (experimental group) and normal HepG2 cells without any treatment were extracted respectively, and their genomic DNA was extracted and used as a template for PCR amplification. After re-annealing, they were treated with T7E1 enzyme and agarose gel electrophoresis was performed. Observe the results of APRF gene knockout.