WO2019237399A1

WO2019237399A1 - Method for specifically knocking out human c2orf40 gene by crispr-cas9 and specific sgrna thereof

Info

Publication number: WO2019237399A1
Application number: PCT/CN2018/091732
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 are a method for specifically knocking out the human C2orf40 gene by CRISPR-Cas9 and a specific sgRNA thereof.

Description

Method for specifically knocking out human C2orf40 gene by CRISPR-Cas9 and specific sgRNA thereof

Technical field

The invention belongs to the technical field of gene editing, and particularly relates to a method for specifically knocking out the human C2orf40 gene by CRISPR-Cas9 and a specific sgRNA thereof.

Background technique

C2orf40 was originally identified and cloned by Bi et al. They found that expression of C2orf40 in esophageal squamous cell carcinoma tissues and cell lines was significantly reduced compared to normal adult esophageal epithelium. Therefore, they believe that C2orf40 may be a new candidate tumor suppressor gene. In recent years, there have been more studies on C2orf40 in esophageal cancer, colon cancer, glioma and prostate cancer.

technical problem

C2orf40 is a candidate tumor suppressor gene for esophageal squamous cell carcinoma (ESCC). It is believed that promoter hypermethylation may be the main mechanism of C2orf40 transcriptional inactivation in ESCC. It is believed that C2orf40 may be involved in regulating the expression of COX-2 by participating in the NF-κB pathway. Taking advantage of its anti-cancer function, it can be used as a potential therapeutic drug for ESCC. It has great potential for transformation and requires a lot of research. However, the lack of a plasmid that enhances the expression of the C2orf40 gene in the prior art has caused certain obstacles to the progress of related research.

Technical solutions

The purpose of the present invention is to provide a method for specifically knocking out the human C2orf40 gene and a specific sgRNA of the CRISPR-Cas9 with simple structure, reasonable design, and convenient use in response to the defects and deficiencies of the prior art. And designed and synthesized a set of sgRNAs that specifically targeted the C2orf40 gene by using CRISPR-Cas9 to specifically knock out the human C2orf40 gene, and ligated the sgRNA with a linear PX330 plasmid to form a vector, and the C2orf40 gene could be achieved by transfecting cells Knockout can achieve a permanent effect; it provides highly efficient sgRNA; sgRNA only needs to synthesize a small amount of polynucleotide fragments, which can be produced in large quantities.

To achieve the above objective, the technical solution adopted by the present invention is:

The CRISPR-Cas9 method for specifically knocking out the human C2orf40 gene and the specific sgRNA thereof according to the present invention adopt the following technical scheme:

1.High-throughput design and selection of sgRNA oligonucleotides

Design sgRNAs via an online design platform (http://crispr-era.stanford.edu/).

2.Constructing oligo double strands of sgRNA

According to the selected sgRNA, add CCGG to its 5 'end to obtain a forward oligonucleotide; according to the selected sgRNA, obtain the complementary strand of its corresponding DNA, and add AAAC to its 5' end to obtain a reverse oligonucleotide . The above-mentioned forward oligonucleotide and reverse oligonucleotide were synthesized separately, and the forward and reverse sequence oligonucleotide sequences of the synthesized sgRNA oligonucleotide were denatured and annealed in pairs. After annealing, the PX330 vector could be ligated. Double-stranded annealing product.

3.Construction of sgRNA oligonucleotide plasmid

① Linearize the PX330 plasmid;

② The annealed sgRNA oligonucleotide double strand was ligated with the linearized PX330 plasmid to obtain the PX330-C2orf40 plasmid.

③ Transform and coat ampicillin plate (100 μg / ml).

④ Sequencing to identify positive clones.

⑤ Shake the bacteria overnight at 37 ℃ and extract the PX330-C2orf40 plasmid without endotoxin.

4.Transfected cells to obtain C2orf40 gene knockout cells

① Follow Lipofectamine 3000 Transfection Reagent (Invitrogen) instruction manual, transfect cells with PX330-C2orf40 vector with corresponding sgRNA oligonucleotides.

②T7EN1 digestion and TA clone sequencing confirmed that the C2orf40 gene had been knocked out.

The invention also provides an sgRNA that specifically targets the C2orf40 gene, the sequence of which is as follows: ID NO. 1.

Beneficial effect

Compared with the prior art, the present invention has the following advantages and effects:

1. The function of the antibody is only a temporary blocking effect. The direct knockout of the C2orf40 gene in the present invention can achieve a permanent effect;

2. There are many types of inhibitory receptors. There is no countermeasure on how to block multiple inhibitory receptors with multiple antibodies. The present invention can be used to knock out multiple coding sequences of C2orf40;

3. The development of effective C2orf40 antibodies is difficult, and the present invention provides a set of highly efficient sgRNAs targeting the human C2orf40 gene;

4. Antibodies can only target extracellular targets, and the present invention can target both extracellular and intracellular targets;

5. The development of antibody drugs is a time-consuming, labor-intensive, and costly process, which makes antibody drugs expensive. Using sgRNA requires only a small amount of synthetic polynucleotide fragments and can be produced in large quantities.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is the structure of the PX330 carrier;

Figure 2 shows the specific cleavage of human sgRNA / Cas9-mediated gene C2orf40 by T7EN1 digestion. Among them, 1- untreated control group 293T cells, 2- experimental group transfected with PX330-C2orf40 vector 293T cells.

Embodiments of the invention

The present invention is described in further detail below with reference to the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

All cell lines used in the examples were purchased from ATCC, PX330 vector was purchased from Addgene, Bbs I enzyme was purchased from Thermo, Endo-Free Plasmid Mini Kit was purchased from Omega-biotek, Lipofectamine 3000 was purchased from Invitrogen, T4 DNA ligase was purchased from NEB, and high-fidelity PCR enzyme was purchased from Promega.

实施例一：靶向人Example 1: Targeting people C2orf40C2orf40 基因的genetic sgRNAsgRNA 寡Widow 聚核苷酸的合成和构建Synthesis and Construction of Polynucleotides

According to the selected sgRNA sequence (SEQ ID No. 1), add CACC to its 5 'end to obtain a forward oligonucleotide; according to the selected sgRNA, obtain its complementary strand, and add AAAC to its 5' end to obtain a reverse oligonucleotide. To the oligonucleotide. The above-mentioned forward oligonucleotide and reverse oligonucleotide were synthesized separately, and the forward and reverse oligonucleotides of the synthesized sgRNA oligonucleotide were denatured and annealed in pairs. After annealing, a double pair that can be ligated into the PX330 vector was formed. Stranded sgRNA oligonucleotide.

The denaturing and annealing system is:

2.5 μL forward oligonucleotide (100 μmol / L)

2.5 μL reverse oligonucleotide (100 μmol / L)

5 μL sterilized ddH2O

Run in the PCR machine according to the following procedures: 95 ° C, 3min; 95-85 ° C, decrease the temperature by 2 ° C / s; 85-25 ° C, decrease the temperature by 0.1 ° C / s; 4 ° hold.

实施例二：利用Example 2: Utilization CRISPR-Cas9CRISPR-Cas9 特异性敲除人Specific knockout C2orf40C2orf40 基因gene

1. Linearize the PX330 plasmid.

The enzyme digestion system and conditions are as follows:

2 μg PX330-C2orf40;

1 μL FastDigest Buffer;

1 μL Bbs I;

Make up ddH2O to 50 μL and incubate at 37 ° C for 1 h.

After the digestion was completed, it was purified by PCR Clean up Kit (Tiangen).

2. The double-stranded sgRNA oligonucleotides that can be ligated into the PX330 vector after denaturation and annealing were ligated with the linearized PX330 plasmid to obtain the PX330-C2orf40 plasmid.

The connection system is as follows:

3 μL, 50 μmol / L purified annealing product

50 μg linearized PX330 plasmid

1 μL 10 × T4 Ligation Buffer

1 μL T4 DNA Ligase (NEB)

ddH2O makes up to 10 μL

Incubate at 16 ° C overnight.

4. Transform the ligated product obtained in the above step into DH5a competent cells and apply ampicillin plates (100 μg / mL), and pick clones.

5. Sequencing method was used to identify positive clones.

6. Cultivate positive clones at 37 ° C with shaker overnight and use Endo-Free Plasmid Mini Kit was extracted from endotoxin-free plasmid extraction kit to obtain PX330-C2orf40 plasmid.

7.Cell culture and transfection

① Inoculate and culture 293T cells with 10% FBS in DMEM medium.

② Divide into 6-well plates before transfection, and perform transfection when the degree of cell fusion reaches 70% -80%.

③ According to Lipofectamine 3000 Transfection Reagent (Invitrogen) instruction manual, transfect 2 μg of PX330-C2orf40 plasmid into each well cell, change the solution after 6-8 hours, and collect the cells after 48 hours.

8. T7EN1 digestion test

① Collect 293T cells transfected with PX330-C2orf40 plasmid and extract genomic DNA.

② Using the extracted genomic DNA as a template, the high-fidelity PCR enzyme was amplified, and the PCR cleanup was purified to obtain the PCR recovered product. 100 ng was uniformly diluted to 20 μL for denaturation and annealing. The procedures were: 95 ° C, 3min; 95-85 ° C, cooling at 2 ° C / s; 85-25 ° C, cooling at 0.1 ° C / s; 4 ° C hold.

③ Add 0.3 μL of T7EN1 to a 20 μL system, and cut for 30 minutes at 37 ° C. Then add 2 μL of 10 × Loading Buffer and test with 1% agarose gel electrophoresis. The results show that the C2orf40 gene was successfully knocked out, as shown in Figure 2. .

Industrial applicability

Claims

The CRISPR-Cas9 method for specifically knocking out the human C2orf40 gene and the sgRNA for specifically targeting the C2orf40 gene are characterized by:

(1) the target sequence of the sgRNA on the C2orf40 gene conforms to the sequence arrangement rule of 5'-GN (20) GG-3 'or 5'-N (21) GG-3';

(2) the target sequence of the sgRNA on the C2orf40 gene is located in the exon of the gene;

(3) The target sequence of the sgRNA on the C2orf40 gene is unique.
The method for specifically knocking out the human C2orf40 gene by CRISPR-Cas9 according to claim 1 and the sgRNA for specifically targeting the C2orf40 gene, wherein the corresponding DNA sequence is shown in SEQ ID NO. 1 of the sequence listing .
The method for specifically knocking out the human C2orf40 gene by CRISPR-Cas9 according to claim 1, wherein:

(1) The sgRNA according to any one of claims 1-2, and CACC is added to the 5 'end of the corresponding DNA sequence to synthesize a forward oligonucleotide; the sgRNA according to any one of claims 1-2 , Obtain the complementary strand of its corresponding DNA sequence, and add AAAC at the 5 ′ end of the complementary strand to obtain a reverse oligonucleotide; synthesize a pair of complementary sgRNA oligonucleotides, the forward and reverse oligonucleotides Acid pair denaturation and annealing to form double-stranded sgRNA oligonucleotides that can be ligated into the PX330 gene editing vector;

(2) The linearized PX330 gene editing vector was used; the annealed double-stranded sgRNA oligonucleotide was linked with the Bbs I linearized PX330 gene editing vector to obtain the PX330-C2orf40 vector, and then the STBL3 competent bacteria were transformed and coated on an ampicillin plate. Positive clones were identified by sequencing; PX330-C2orf40 vector was extracted without shaking the positive clones at 37 ° C with a shaker overnight;

(3) PX330-C2orf40 vector was mixed with liposomes to transfect cells;

(4) T7EN1 digestion test confirmed that the C2orf40 gene had been knocked out and knocked out cells were obtained.
The method for specifically knocking out the human C2orf40 gene by CRISPR-Cas9 according to claim 3, wherein the liposome in step (3) is Lipofectamine 3000 (Invitrogen).