WO2019222972A1

WO2019222972A1 - Crispr/cas9 targeted knockout human gi24 gene and specificity grna thereof

Info

Publication number: WO2019222972A1
Application number: PCT/CN2018/088284
Authority: WO
Inventors: 毛吉炎
Original assignee: 深圳市博奥康生物科技有限公司
Priority date: 2018-05-24
Filing date: 2018-05-24
Publication date: 2019-11-28

Abstract

On the basis of a design principle for CRISPR/Cas9 gRNA, two target sites are designed on human GI24 gene, a corresponding oligonucleotide is synthesized, and same is constructed on carrier px459. A CRISPR/Cas9 system guided by utilizing the recombinant carrier in Jurkat cells allows the effective knockout of human GI24 gene.

Description

CRISPR / Cas9 targeted knockout of human GI24 gene and its specific gRNA

Technical field

The invention belongs to the field of genetic engineering and biomedical technology, and particularly relates to a CRISPR / Cas9 targeted knockout human GI24 gene and a specific gRNA thereof.

Background technique

The GI24 protein is a new member of the PD-L1 protein family. It is a novel and differently structured Ig superfamily inhibitory ligand. Its ectodomain has homology to the B7 family ligand PD-L1.

technical problem

Existing studies have found that GI24 protein plays an important role in the cellular immunity of a variety of cancers and autoimmune diseases, allergies, infections and inflammatory conditions such as multiple sclerosis and joint disorders. A large number of translational studies are required before it can be applied to the clinic. There is a lack of methods for targeted knockout of GI24 gene expression in some technologies, which has caused certain obstacles to the progress of related research.

Technical solutions

In view of the above problems, the present invention provides a CRISPR / Cas9 targeted knockout of the human GI24 gene and its specific gRNA. The gRNA sequence can be used to knock out the human GI24 gene, thereby inhibiting or eliminating the expression of GI24.

The technical solution of the present application is as follows:

1. Design and synthesis of highly efficient gRNA targeting human GI24 gene and construction of gRNA / cas9 expression system.

2. Analyze and detect the inhibitory effect of the gRNA-guided CRISPR / Cas9 system of the present invention on the GI24 gene in Jurkat cells.

Beneficial effect

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

The invention designs and synthesizes two single-stranded oligo sequences according to the gRNA guide sequence, anneals to form a double strand, and then connects with the Cas9 vector. The Cas9 vector is used to introduce the gRNA and CRISPR system into the target cell. The Cas9 protein will find a match under the guidance of the gRNA. The DNA sequence was cut to achieve the GI24 gene knockout.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the Western Blot results of Jurkat cells in the control and experimental groups.

Embodiments of the invention

All cell lines used in the examples were purchased from ATCC, px459 vector was purchased from Addgene, endonuclease Bbs I was purchased from Thermo, Endo-Free Plasmid Mini Kit was purchased from Omega-biotek, Lipofectamine 2000 was purchased from Invitrogen, T4 DNA ligase Commercially available from NEB and puromycin from Sigma.

实施例一Example one 靶向Target GI24GI24 基因的genetic gRNAgRNA 设计design

The human GI24 gene sequence was found in GenBank, and potential target sites were designed in the exon region of the human GI24 gene. Using online design tools and gRNA design principles, evaluate the target site with high score on the human GI24 gene sequence to design gRNA, whose sequence is shown in SEQ ID NO.1, and then add CACC to its 5 'end to obtain a positive oligo. Nucleotides, and add AAAC to the 5 'end of its reverse complement. The above-mentioned forward oligonucleotide and reverse oligonucleotide were synthesized separately, denatured at 95 ° C, and annealed to form a double-stranded DNA molecule that can be ligated into the px459 vector.

实施例二Example two 构建表达Construct expression gRNAgRNA 的载体Carrier

The px459 vector was treated with Bbs I enzyme at 37 ° C for 1 h, and then electrophoresed on 1% agarose to recover the digested product. The double-stranded DNA molecule that can be ligated into the px459 vector obtained in Example 1 was ligated with the px459 vector using T4 DNA ligase. The ligation system (10 μl) was: annealed double-stranded (GI24-gRNA) 2 μl, px459 vector 2 μl, 10 × T4 DNA Ligase Buffer 1 μl, T4 DNA Ligase 1 μl, ddH2O made up to 10 μl; connection conditions: 16 ° C overnight.

The ligation product is transformed into competent cells Stbl3. The specific transformation method is: take out the competent cells Stbl3 at -80 ° C, and dissolve them in an ice bath; then take 1 μl of the above-mentioned ligation products to 50 μl of competent cells and mix for 30 minutes on ice 42 ℃ water bath for 60 s, do not shake during the process; cool in ice bath for 2 min; then add 800 μl LB medium, shake at 37 ° C for 30 min; spread on LB plates containing 100 μg / ml ampicillin and culture overnight. Pick positive clones and shake at 37 ° C overnight for expansion and sequencing. The correct sequencing is the required Cas9 vector to target the GI24 gene, named px459-GI24 vector.

实施例三Example three 无内毒素质粒Endotoxin-free plasmid DNADNA 的制备Preparation

The correct strain was sequenced and identified in Example 2 and placed in an LB liquid medium having an ampicillin concentration of 100 μg / ml, and cultured at 250 rpm and 37 ° C. with shaking for 12-16 hours. Collect the bacterial solution by centrifugation at 10,000 rpm at 4 ° C, discard the supernatant, collect the bacterial cells, and then extract the plasmid according to the instructions of the Endo-Free Plasmid Mini Kit kit to obtain the px459-GI24 vector without endotoxin.

Example 4 Transfection of Jurkat Cells

Three days before transfection, Jurkat cells were resuscitated. The cells were placed in a 10% FBS + DMEM culture flask and cultured in a 37 ° C, 5% CO2 incubator. One day before transfection, the recovered cells were subcultured.

Absorb the culture medium from the T75 flask of cultured Jurkat cells, add 2 ml of 0.25% trypsin taken out from the refrigerator at 4 ° C to cover the bottom of the bottle uniformly, place in a 37 ° C incubator for 3-5 minutes, remove and shake to find The cells are detached at the bottom, shake them all down, add 3 ml of pre-heated 10% DMEM in a 37 ° C water bath, and pipette with a 10 ml pipette for 6-8 times, leaving no dead corners. The bottle mouth is more difficult to pipette. The pipette can be pointed at the mouth of the culture tube, and the medium close to the bottle mouth can be covered by punching the medium with a small force. After that, aspirate all the cells and place them in a 15 ml centrifuge tube. Take 50ul of the mixed cells into a 1.5ml EP tube, add 450 μl of 10% DMEM, which is a 10-fold dilution, mix and take 10 μl of cells in Count in the counting board. The day of passage is recorded as the first day. If transfection is performed the next day, spread 9-10 × 10 ⁷ / T75; if transfection is performed on the third day, spread 3.5-4 × 10 ⁷ / T75. Add 15 ml of 10% DMEM medium to each T75 bottle. Cell density was observed on the day of transfection, and the degree of fusion reached 80-90%. Follow the instructions in Lipofectamine 2000 instructions to transfect px459-GI24 into Jurkat cells.

48 hours after transfection, trypsinize the adherent cells after transfection, collect the cells by centrifugation, remove the waste liquid and add 1 ml of PBS to resuspend the cells. Take 500 μl into the original bottle to continue the culture, and put the remaining cells into 1 .5ml centrifuge tube to extract total protein.

实施例五Example 5 Western Blot Western Blot 检测转染效果Detection of transfection effect

Jurkat cells without any treatment were used as the control group, and the cells obtained in Example 4 were used as the experimental group. 100-200 μl of 5 × SDS-PAGE loading buffer was added, and the mixture was boiled in boiling water for 5 minutes. 15 μl of the loaded SDS- PAGE protein electrophoresis. After electrophoresis, semi-dry transfer with conventional proteins, blocking with 10% skim milk powder for 2 h, put the blocked PVDF membrane in rabbit anti-human GI24 antibody, rinse the buffer 3 times, and then transfer the membrane to goat anti-rabbit secondary antibody Buffer, incubate at room temperature for 60 min, and rinse 4 times with rinsing buffer. After the rinsing was completed, the Western blot membrane was developed and detected by ECL, and the results are shown in FIG. 1. It can be seen that the GI24 protein band cannot be detected by Western Blot in the GI24 frameshift mutation Jurkat cells, while the GI24 protein band appears in the control group, indicating that the gRNA sequence used to knock out the GI24 gene of human cells can achieve GI24. Gene knockout.

Industrial applicability

Claims

A gRNA for specifically targeting the human GI24 gene in a CRISPR / Cas9-specific knockout human GI24 gene is characterized in that the target sequence of the gRNA on the human GI24 gene conforms to 5'-N (20) -NGG3 ' Or the sequence of 5'-CCN-N (20) -3 'is regular, and the target sequence on the human GI24 gene is unique.
The gRNA for specifically targeting the human GI24 gene in the CRISPR / Cas9-specific knockout human GI24 gene according to claim 1, wherein the targeting site is as shown in SEQ ID NO .1 shown.
The gRNA for specifically targeting the human GI24 gene in the CRISPR / Cas9 specific knockout human GI24 gene according to claim 2, wherein the tumor cell is a Jurkat cell.