WO2019237397A1

WO2019237397A1 - Method for knocking out human pin1 gene

Info

Publication number: WO2019237397A1
Application number: PCT/CN2018/091727
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

Provided is a method for knocking out a human PIN1 gene by using a CRISPR-Cas9 gene editing technology. Specific operation steps comprise: (1) designing an sgRNA sequence; (2) performing connection, conversion and amplification of sgRNA; (3) performing plasmid transfection on a 293T cell and packaging same to lentivirus; (4) performing lentivirus infection on a target cell and performing puromycin screening; and (5) performing verification on a PIN1 gene knockout result. Experiments prove that the provided method for knocking out the PIN1 gene can efficiently knock out the PIN1 gene of a cell, and can serve as a powerful tool to be applied to PIN1-related drug research and development.

Description

Method for knocking out human PIN1 gene

Technical field

The invention mainly relates to the field of genetic engineering, in particular to a method for knocking human PIN1 gene by using CRISPR-Cas9 gene editing technology.

Background technique

Serine / threonine phosphorylation modification in proteins is involved in regulating a variety of signal transduction pathways and is involved in maintaining normal life activities of cells. Peptidyl-prolyl isomerase 1 1. PIN1) The spatial structure of silk / threonine-proline that can specifically catalyze phosphorylation occurs, so the conformation of some oncoproteins with specific silk / threonine-proline motifs can be changed, leading to oncoproteins Can not be degraded by the ubiquitination process, oncoprotein accumulation, and promote tumorigenesis.

technical problem

Studies have shown that PIN1 participates in the regulation of ubiquitination as a new ubiquitination conversion mechanism. It belongs to one of the members of the polypeptide prolyl cis-trans isomerase family, and its physiological characteristics are composed of two functional domains: the carboxy-terminal peptide Prolyl cis-trans isomerase (PPlase active region) and the tryptophan-tryptophan central region at the amino terminus. PIN1 can change the conformation of the protein, thereby changing the spatial structure of the substrate, affecting the affinity between the substrate and ubiquitin ligase, leading to abnormal protein degradation, resulting in the oncoprotein cannot be degraded by the ubiquitination process, and the accumulation of oncoproteins, Involved in the development of tumors. Because of this characteristic, PIN1 participates in the regulation of many cyclins, and thus in the regulation of cell proliferation and differentiation. It plays an important role in the process of tumor formation and can be used as a therapeutic target for clinical transformation, but it is lacking in the existing technology. The method of specifically knocking out the human PIN1 gene has caused certain obstacles 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 the human PIN1 gene by using CRISPR-Cas9 gene editing technology. The specific operation steps are as follows:

(1) Design the sgRNA sequence

In the region of the PIN1 gene expression DNA that is close to 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 PIN1 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 passages, transfection was performed: CRISPR-Cas9 system containing the knock-out PIN1 gene expression plasmid and two helper plasmids each 1 μg were transfected to 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 Hela 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. After 7-14 days, the cells infected with lentivirus will form single cell clones, and the cell selection is completed.

(5) PIN1 gene knockout result verification

The selected Hela cells (experimental group) and normal Hela cells without any treatment were taken, and their genomic DNA was extracted and PCR-amplified using the template as a template. After re-annealing, they were treated with T7E1 enzyme and agarose gel electrophoresis was performed. Observe the results of PIN1 gene knockout.

Beneficial effect

The PIN1 gene knockout method 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 PIN1 gene, and can be used in research and development of drugs related to abnormal PIN1 expression.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing the results of identifying the editing status of the PIN1 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 region of the PIN1 gene expression DNA that is close to 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’- GCAGAGCGCGTCTAGCCAAT -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 ' - CACCGCAGAGCGCGTCTAGCCAAT -3 ’and 5’- AAACATTGGCTAGACGCGCTCTGC -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-PIN1 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 operation was performed: pLenti CRISPR-PIN1 plasmid and two lentiviral packaging helper plasmids each were taken 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: HelaHela 细胞的慢病毒感染及嘌呤霉素筛选Lentiviral infection of cells and puromycin selection

Culture Hela cells. When the cell confluency 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 PIN1PIN1 基因敲除结果Knockout results

The Hela cells (experimental group) and normal Hela cells (control group) infected with lentivirus were expanded and cultured, and their 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 PIN1 gene in Hela cells was successfully edited.

Industrial applicability

Claims

A method for knocking out the human PIN1 gene, characterized in that the method includes the following operation steps:

(1) Design the sgRNA sequence

In the PIN1 gene expression DNA region near the N-terminus of the protein coding region, find the sequence that begins 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 PIN1 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 passages, transfection was performed: CRISPR-Cas9 system containing the knock-out PIN1 gene expression plasmid and two helper plasmids each 1 μg were transfected to 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 the cultured Hela 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) and treat for 24 h. 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) PIN1 gene knockout result verification

The selected Hela cells (experimental group) and normal Hela cells without any treatment were taken, and their genomic DNA was extracted and PCR-amplified using the template as a template. After re-annealing, they were treated with T7E1 enzyme and agarose gel electrophoresis Observe the results of PIN1 gene knockout.