WO2019237386A1

WO2019237386A1 - Method for knocking out human ct1.1 gene

Info

Publication number: WO2019237386A1
Application number: PCT/CN2018/091716
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 the human CT1.1 gene by using CRISPR-Cas9 gene editing technology, the method comprising the following specific operation steps: (1) design of an 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 with puromycin, and (5) verification of the CT1.1 gene knockout result. The method can be applied to drug research and development associated with CT1.1.

Description

Method for knocking out human CT1.1 gene

Technical field

The invention mainly relates to the field of genetic engineering, and in particular, to a method for knocking human CT1.1 gene by using CRISPR-Cas9 gene editing technology.

Background technique

Cancer / testis antigen (CTA) is a group of tumor-associated antigens. It is not expressed in normal human tissues except testis, placenta, and trophoblast cells, but is highly expressed in a variety of tumor tissues, making it a recent year. Tumor immunotherapeutic target antigen has been widely concerned. Melanin-associated antigen (MAGE) is a class of tumor-associated antigens isolated from melanoma cells. It is a subfamily of CTA. It can be processed into antigen peptides in tumor cells, combined with human leukocyte antigen Ⅰ, and then Autocytotoxic T lymphocytes recognize and kill tumor cells.

technical problem

CT1.1 is the earliest found member of the MAGE family. It is specifically expressed in malignant tumors and can bind to MHC molecules. It can be recognized by specific cell T-cell receptors and activated into cytotoxic T-lymph with specific T-cell receptors. Cells enable the body to produce an immune response that is dominated by cellular immunity. Therefore, CT1.1 is a tumor-specific ideal target antigen, which has become a hot topic in recent years. However, the lack of a specific method for human CT1.1 gene deletion in the prior art 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 CT1.1 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 CT1.1 gene expression DNA 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 up and cultured, and then a large number of plasmids were extracted to obtain a constructed CRISPR-Cas9 containing the knock-out CT1.1 gene System expression plasmid, save for future 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: 1 μg each of the CRISPR-Cas9 system expression plasmid containing the CT1.1 gene knockout and 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 and culture A375 cells. When the cell confluency 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 CT1.1 gene knockout results

The screened A375 cells (experimental group) and normal A375 cells without any treatment were taken, respectively, and genomic DNA was extracted and used as a template for PCR amplification. After reannealing, treatment with T7E1 enzyme and agarose gel electrophoresis Observe the results of CT1.1 gene knockout.

Beneficial effect

The method for knocking out the CT1.1 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 CT1.1 gene, and can be used in research and development of drugs related to abnormal CT1.1 expression.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the results of identifying the CT1.1 gene editing status 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 CT1.1 gene expression DNA 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’- GAGGTTTCCATTCTGAGGGA -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 ' -CACCGAGGTTTCCATTCTGAGGGA -3 'and 5'-AAACTCCCTCAGAATGGAAACCTC -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-CT1.1 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 in growth culture before transfection: take pLentiCRISPR-CT1.1 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: A375A375 细胞的慢病毒感染及嘌呤霉素筛选Lentiviral infection of cells and puromycin selection

Culture A375 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 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 CT1.1CT1.1 基因敲除结果Knockout results

A375 cells (experimental group) and normal A375 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 CT1.1 gene in A375 cells was successfully edited.

Industrial applicability

Claims

A method for knocking out the human CT1.1 gene, wherein the method includes the following operation steps:

(1) Design the sgRNA sequence

In the part of the CT1.1 gene expression DNA 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 with the sequence of other genes or The homology 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 up and cultured, and then a large number of plasmids were extracted to obtain a constructed CRISPR-Cas9 containing the knock-out CT1.1 gene. System expression plasmid, save for future 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: 1 μg each of the CRISPR-Cas9 system expression plasmid containing the CT1.1 knockout gene and two helper plasmids were cotransfected with Lipofectamine 3000 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 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 and culture A375 cells and pass through the growth culture twice. 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) Validation of CT1.1 gene knockout results

The screened A375 cells (experimental group) and normal A375 cells without any treatment were taken, respectively, and genomic DNA was extracted and used as a template for PCR amplification. After reannealing, the cells were treated with T7E1 enzyme and agarose gel electrophoresis was performed. Observe the results of CT1.1 gene knockout.