WO2019222971A1 - Crispr/cas9-targeted knockout of human vstm3 gene and specific grna therefor - Google Patents

Abstract

Provided is specific gRNA for targeted knockout of human VSTM3 gene. According to the design principle of CRISPR/Cas9 gRNA, two target sites are designed on human VSTM3 gene, and corresponding oligonucleotide is synthesized and then constructed on px459 vector. Under the guidance of the provided gRNA, a CRISPR/Cas9 system is expected to be applied in new-type drugs for treating tumors.

Description

CRISPR / Cas9 targeted knockout of human VSTM3 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 VSTM3 gene and a specific gRNA thereof.

Background technique

VSTM3 is a newly discovered membrane protein molecule that is widely expressed on the surface of Treg cells, memory T cells, activated T cells, and NK cells. The study found that: ① VSTM3-Fc fusion protein can promote DC secretion of IL-10 and reduce proinflammatory factor IL-12; ② VSTM3 protein in vivo can significantly reduce late-onset hypersensitivity, and its effect is similar to that of CTLA-4 protein Equivalent; ③ The ability of mature DC treated with VSTM3-Fc fusion protein to stimulate T cell proliferation is weakened, which is related to DC secretion of IL-10. Increasing evidence shows that VSTM3 is an important immunomodulator, similar in function to ITIM, which can inhibit the cytotoxicity of NK cells, the activation and proliferation of CD4 + T cells, and the production of cytokines.

technical problem

VSTM3 is a substance that inhibits the function of T cells. It plays a role in inhibiting the excessive activation of T cells under normal conditions; however, this feature also makes it easy for tumor cells to use it to achieve immune escape. Therefore, VSTM3 is a potential target for tumor therapy and requires in-depth research. However, the lack of a means to target VSTM3 gene expression in the prior art 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 VSTM3 gene and a specific gRNA thereof. The gRNA sequence can be used to knock out the human VSTM3 gene, thereby suppressing or eliminating VSTM3 expression.

The technical solution of the present application is as follows:

1. Design and synthesis of highly efficient gRNA targeting human VSTM3 gene and construction of gRNA / cas9 expression system.

2. Analyze and detect the inhibitory effect of the gRNA-guided CRISPR / Cas9 system on VSTM3 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 VSTM3 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

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, 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.

Example one Target VSTM3 genetic gRNA design

The sequence of the human VSTM3 gene was found in GenBank, and potential target sites were designed in the exon region of the human VSTM3 gene. Using online design tools and gRNA design principles, evaluate the high-scoring target site on the human VSTM3 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.

Example two Construct expression gRNA 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 (VSTM3-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; Do not shake during 42 s water bath for 60 s; cool in ice bath for 2 min; then add 800 μl LB medium and shake at 37 ℃ for 30 min; LB plate coated with 100 μg / ml ampicillin and culture overnight. After picking positive clones Shake at 37 ° C overnight for expansion and send for sequencing. The correct sequencing is the required Cas9 vector targeting VSTM3 gene, named px459-VSTM3 vector.

Example three Endotoxin-free plasmid DNA 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 endotoxin-free px459-VSTM3 vector.

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%. Transfect px459-VSTM3 into Jurkat cells according to the instructions of Lipofectamine 2000.

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.

Example 5  Western Blot 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 VSTM3 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 VSTM3 protein bands are not detected in Western Blot in VSTM3 frameshift gene mutation Jurkat cells, while VSTM3 protein bands appear in the control group, indicating that the gRNA sequence used to knock out the VSTM3 gene of human cells can achieve VSTM3 Gene knockout.

Industrial applicability

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 VSTM3 gene knockout.

Claims (3)

1. A gRNA for specifically targeting the human VSTM3 gene in a CRISPR / Cas9-specific knockout human VSTM3 gene is characterized in that the target sequence of the gRNA on the human VSTM3 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 VSTM3 gene is unique.
2. The gRNA for specifically targeting the human VSTM3 gene in the CRISPR / Cas9-specific knockout human VSTM3 gene according to claim 1, wherein the targeting site is as shown in SEQ ID NO .1 shown.
3. The gRNA for specifically targeting the human VSTM3 gene in the CRISPR / Cas9 specific knockout human VSTM3 gene according to claim 2, wherein the tumor cell is a Jurkat cell.