WO2019237390A1

WO2019237390A1 - Method for knocking out human hdmx gene

Info

Publication number: WO2019237390A1
Application number: PCT/CN2018/091720
Authority: WO
Inventors: 毛吉炎
Original assignee: 深圳市博奥康生物科技有限公司
Priority date: 2018-06-16
Filing date: 2018-06-16
Publication date: 2019-12-19

Abstract

A method for knocking out a human HDMX gene by using CRISPR-Cas9 gene editing technology, comprising: (1) designing an sgRNA sequence; (2) ligating, transforming and amplifying the sgRNA; (3) a transfecting 293T cell with a plasmid and packaging into a lentivirus; (4) infecting a target cell with the lentivirus and screening using puromycin; (5) verifying an HDMX gene knockout result. In the method, an HDMX gene of a cell may be knocked out and may be used for drug research and development related to HDMX.

Description

Method for knocking out human HDMX gene

Technical field

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

Background technique

The p53 tumor suppressor gene is located on the short arm of human chromosome 17 and encodes a protein containing 393 amino acids, namely the p53 protein. Wild-type p53 protein can be used as a cell cycle regulator protein to monitor damage and induce apoptosis, inhibit excessive cell proliferation, and have anti-tumor cell proliferation functions. Once the p53 gene is mutated, due to changes in its spatial conformation, it loses its regulatory effect on cell growth, apoptosis, and repair. A variety of target genes of the p53 gene mainly include: P21, HDMX, bax, etc. It is these boot genes regulated by the p53 gene and form a network to achieve the functions jointly. Among them, HDMX-p53-p21 signaling pathway is one of the important pathways in the gene pathway.

The human HDMX gene encodes a protein with a molecular weight of kD. There are two promoters, P1 is upstream of the coding gene, and P2 is in the first intron, which is controlled by p53 through two nearby p53 binding sites. HDMX-encoded protein is divided into 4 functional regions: about 10 amino acid residues at the N-terminus, which can be combined with p53. This region also has a nuclear localization sequence and a highly acidic region of nuclear export signals; a zinc finger structure that can bind to genes and activate genes To make cells from G1 phase to S phase; ring finger structure, which can mediate protein-protein interactions, participate in cell regulation and promote cell proliferation.

technical problem

Studies have shown that HDMX negatively regulates p53 through multiple pathways. HDMX protein can directly bind to p53 to inhibit its activity, resulting in p53 being degraded by the ubiquitin system. Different splicing forms of HDMX also participate in regulatory activity. In a variety of tumors, the abnormal amplification of HDMX or the increase in protein expression levels lead to the inactivation of p53 function, so it is considered to be a newly discovered important proto-oncogene. In addition, HDMX protein has been confirmed to be closely related to the degree of tumor metastasis. In tumor metastasis and recurrence, high amplification of HDMX gene or high expression of HDMX protein were observed. Therefore, HDMX is an ideal target for potential tumor treatment. However, the lack of a method for specifically knocking out the human HDMX gene 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 HDMX gene by using CRISPR-Cas9 gene editing technology. The specific operation steps are as follows:

(1) Design the sgRNA sequence

In the expression DNA region of the HDMX gene, 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, such as 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

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

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 HDMX 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: 1 μg each of the expression plasmid containing the CRISPR-Cas9 system with the knockout HDMX gene and two helper plasmids were co-transfected 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 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 and culture MCF-7 cells. When the confluency 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 lentiviral solution 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 HDMX gene knockout results

Take the selected MCF-7 cells (experimental group) and normal MCF-7 cells without any treatment, extract their genomic DNA, and use them as templates for PCR amplification. After re-annealing, treat with T7E1 enzyme, agar Glucose gel electrophoresis was used to observe the results of HDMX gene knockout.

Beneficial effect

The HDMX gene knockout method provided by the present invention and a cell line constructed by using the method provide an experimental technology platform for further exploring the role of the HDMX gene, and can be used in research and development of drugs related to abnormal HDMX expression.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the results of identification of the editing status of HDMX genes 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 expression DNA region of the HDMX gene, 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’- GTGAAACTGTTAGAGCCTTT -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 ' - CACCGTGAAACTGTTAGAGCCTTT -3 ’and 5’- AAACAAAGGCTCTAACAGTTTCAC -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 is used for expansion culture, and then the endotoxin-free plasmid extraction kit is used to extract the recombinant vector therein, and a large number of constructed CRISPR-Cas9 system-containing expression vectors pLentiCRISPR-HDMX are obtained.

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

The 293T cells were thawed and cultured, and the cells were transfected twice after growth and culture. The transfection was performed: pLenti CRISPR-HDMX plasmid and two lentiviral packaging helper plasmids were taken each 1 μg, and co-transfected into 293T cells with Lipofectamine 3000. 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: MCF-7MCF-7 细胞的慢病毒感染及嘌呤霉素筛选Lentiviral infection of cells and puromycin selection

Culture MCF-7 cells. When the confluency 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 lentiviral solution was changed to a complete medium containing 1 μg / mL puromycin, and the screening culture was started. The screening time was 7-14 days. 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 HDMXHDMX 基因敲除结果Knockout results

The MCF-7 cells infected with lentivirus (experimental group) and normal MCF-7 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 HDMX gene in MCF-7 cells was successfully edited.

Industrial applicability

Claims

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

(1) Design the sgRNA sequence

In the part of the HDMX 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 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 HDMX 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: 1 μg each of the expression plasmid containing the CRISPR-Cas9 system with the knockout HDMX gene and two helper plasmids were co-transfected 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

MCF-7 cells were thawed and cultured for two passages. When the confluence of the cells was about 70% -80%, a mixed solution of lentivirus and culture medium (containing 4 μg / mL polybrene) was added for 24 h. The virus solution 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 HDMX gene knockout results

Take the selected MCF-7 cells (experimental group) and normal MCF-7 cells without any treatment, extract their genomic DNA, and use them as templates for PCR amplification. After re-annealing, treat with T7E1 enzyme, agar Glucose gel electrophoresis was used to observe the results of HDMX gene knockout.