WO2019222970A1 - Crispr/cas9 targeted knockdown of human cd226 gene and specific grna thereof - Google Patents

Abstract

Provided is a specific gRNA for the targeted knocking down of a human CD226 gene, wherein two target sites are designed on the human CD226 gene according to the design principle of CRISPR/Cas9 gRNA, and a corresponding oligomeric nucleotide is synthesized and then constructed on a px459 vector. The human CD226 gene may be effectively knocked out using a recombinant vector-directed CRISPR/Cas9 system in Jurkat cells. The provided gRNA-directed CRISPR/Cas9 system is expected to acquire applications in new drugs for treating tumors.

Description

CRISPR / Cas9 targeted knockout of human CD226 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 CD226 gene and a specific gRNA thereof.

Background technique

CD226 molecule is a widely expressed immune system such as T cells, NK cells, NK T cells, activated vascular endothelial cells, megakaryocyte / platelet lineage, monocytes, thymocytes, certain B cell subsets, some hematopoietic stem cells and mast cells. Cell surface type I transmembrane glycoprotein.

technical problem

The interaction of CD226 and its ligands participates in the activation, differentiation and cytotoxicity of CTL and NK cells, regulates the proliferation and differentiation of CD4 + T cells, mediates the adhesion of endothelial cells and other cells, promotes the activation and aggregation of platelets, and participates in hematopoietic regulation Mediates the degranulation of mast cells, participates in the anti-apoptotic effects of NKT cells and thymocytes, the maturation of dendritic cells, the formation of immune synapses and synapses, and has important potential in immunotherapy of tumors. Intensive translational research has been conducted, but the lack of means for targeted knock-out of CD226 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 human CD226 gene and its specific gRNA. The gRNA sequence can be used to knock out the human CD226 gene, thereby suppressing or eliminating the expression of CD226.

The technical solution of the present application is as follows:

1. Design and synthesis of high-efficiency gRNA targeting human CD226 gene and construction of gRNA / cas9 expression system.

2. Analyze and detect the inhibitory effect of the gRNA-guided CRISPR / Cas9 system on the CD226 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 CD226 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.

Example one Target CD226 genetic gRNA design

The sequence of human CD226 gene was found in GenBank, and potential target sites were designed in the exon region of human CD226 gene. By using online design tools and gRNA design principles, we evaluated the high-scoring target sites on the human CD226 gene sequence to design the gRNA. The sequence is shown in SEQ ID NO.1, and then the CACC was added to the 5 ′ end to obtain the 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 (CD226-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 was the Cas9 vector required to target the CD226 gene, and was named px459-CD226 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 px459-CD226 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%. Transfect Jurkat cells with px459-CD226 according to Lipofectamine 2000 instructions.

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 protein, blocking with 10% skimmed milk powder for 2 h, place the blocked PVDF membrane in rabbit anti-human CD226 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 CD226 protein band cannot be detected by Western Blot in CD226 frameshift gene mutation Jurkat cells, while the CD226 protein band appears in the control group, indicating that the gRNA sequence used for knocking out the CD226 gene of human cells can achieve CD226 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 CD226 gene knockout.

Claims (3)

A gRNA for specifically targeting the human CD226 gene in a CRISPR / Cas9-specific knockout human CD226 gene, characterized in that the target sequence of the gRNA on the human CD226 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 CD226 gene is unique. The gRNA for specifically targeting the human CD226 gene in the CRISPR / Cas9-specific knockout human CD226 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 CD226 gene in the CRISPR / Cas9 specific knockout human CD226 gene according to claim 2, wherein the tumor cell is a Jurkat cell.