WO2019210491A1

WO2019210491A1 - Crispr/cas9 targeted knockout of human cvap gene and specific grna thereof

Info

Publication number: WO2019210491A1
Application number: PCT/CN2018/085518
Authority: WO
Inventors: 毛吉炎
Original assignee: 深圳市博奥康生物科技有限公司
Priority date: 2018-05-03
Filing date: 2018-05-03
Publication date: 2019-11-07

Abstract

Provided is a gRNA for specifically targeting the human CVAP gene during the specific knockout of the human CVAP gene with CRISPR/Cas9.

Description

CRISPR/Cas9 Targeted Knockout Human CVAP 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 CVAP gene and a specific gRNA thereof.

Background technique

Alzheimer's disease (AD) is the most common cause of senile dementia. This disease is common in the elderly. It is a central system degenerative degenerative disease characterized by progressive cognitive impairment and memory impairment. The clinical manifestations are deteriorating cognitive and memory functions, progressive decline in daily living ability, and various Psychiatric symptoms and behavioral disorders. More onset in the old age, latent onset, slow and irreversible, clinically based on intelligent damage. CVAP is a single transmembrane protein with a membrane receptor-like protein structure that is widely present in various tissues of the body. Β-amyloid (Aβ) is a fragment containing 39 to 43 amino acids which is cleaved by β and γ secretase in CVAP under pathological conditions. The relative molecular mass is about 4.2×103, and it is deposited outside the cell. The gap forms age spots.

technical problem

At present, AD has become the fourth major disease causing human death, seriously affecting the physical and mental health and quality of life of the elderly. In the susceptible population, Alzheimer's disease may cause many family social problems, and study CVAP in Alzheimer's disease. The role of the disease process has great significance for the early prevention and treatment of symptomatic Alzheimer's disease, but the lack of targeted means of knocking out CVAP gene expression in the prior art has hindered the progress of related research. .

Technical solution

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

The technical solution of the application of the present invention is as follows:

1. High-efficiency gRNA design and synthesis targeting human CVAP gene and construction of gRNA/cas9 expression system.

2. Analysis of the inhibitory effect of the gRNA-directed CRISPR/Cas9 system of the present invention on the CVAP gene in RGC5 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 targeting sequence, anneals to form a double strand, and then connects with the Cas9 vector, and uses the Cas9 vector to introduce the gRNA and the CRISPR system into the target cell, and the Cas9 protein is found to be matched under the guidance of the gRNA. The DNA sequence was cut to achieve knockout of the CVAP gene.

DRAWINGS

Figure 1 is a graph showing the results of Western Blot of RGC5 cells in the control and experimental groups.

Embodiments of the invention

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

实施例一靶向Example 1 Targeting CVAPCVAP 基因的genetic gRNAgRNA 设计design

The sequence of the human CVAP gene was found in GenBank, and potential target sites were designed in the exon region of the human CVAP gene. The gRNA was designed to evaluate the target sites with higher scores on the human CVAP gene sequence by online design tools and gRNA design principles. The sequence is shown in SEQ ID NO. 1, and then CACC is added to the 5' end to obtain a positive oligo. Nucleotide, and AAAC is added to the 5' end of its reverse complement. The above forward oligonucleotide and reverse oligonucleotide were separately synthesized, denatured at 95 ° C, and annealed to form a double-stranded DNA molecule ligated into the px459 vector.

实施例二构建表达Example 2 constructs expression gRNAgRNA 的载体a

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 ligated into the px459 vector obtained in Example 1 was ligated with the px459 vector using T4 DNA ligase. The ligation system (10 μl) is: annealed double strand (CVAP-gRNA) 2 μl, px459 vector 2 μl, 10 × T4 DNA Ligase Buffer 1 μl, T4 DNA Ligase 1 μl, ddH2O was made up to 10 μl; ligation conditions: overnight at 16 °C.

The ligation product was transformed into competent cell Stbl3, and the specific transformation method was as follows: -Compatible cell Stbl3 was taken out at -80 ° C, and dissolved in an ice bath; then, 1 μl of the above-mentioned ligation product was added to 50 μl of competent cells, and the mixture was mixed and ice bathed for 30 min; 42 ° C water bath for 60 s, do not shake during the process; ice bath cooling for 2 min; then add 800 μl LB medium, shaken at 37 ° C for 30 min; coated with 100 μg / ml ampicillin in LB plate overnight, pick positive clones The shaker was shaken overnight at 37 ° C for expansion and sequencing. The correct sequencing is the desired Cas9 vector targeting the CVAP gene, designated px459-CVAP vector.

实施例三无内毒素质粒Example 3 Endotoxin-free plasmid DNADNA 的制备Preparation

The correct strain was identified by sequencing in Example 2, and placed in an LB liquid medium with an ampicillin concentration of 100 μg/ml, and shake cultured at 250 rpm and 37 ° C for 12-16 h. The bacterial solution was collected by centrifugation at 10,000 rpm at 4 ° C, the supernatant was discarded, and the cells were collected, and then the plasmid was extracted according to the procedure of the Endo-Free Plasmid Mini Kit kit to obtain an endotoxin-free px459-CVAP vector.

Example 4 Transfection of RGC5 cells

Three days before transfection, RGC5 cells were resuscitated, and the cells were placed in a 10% FBS+DMEM flask and cultured in a 37 ° C, 5% CO 2 incubator. The day before transfection, the resuscitated cells were subcultured.

The medium in the T75 flask of the cultured RGC5 cells was sucked up, and 0.25% trypsin taken out in a 2 ml 4 ° C refrigerator was added to uniformly cover the bottom of the bottle, placed in a 37 ° C incubator for 3-5 min, taken out, and shaken to be found. The cells were detached at the bottom, and all of them were shaken off. Add 10 ml of DMEM preheated in a 3 ml 37 ° C water bath, blow with a 10 ml pipette, and blow 6-8 times without leaving a dead angle. It is difficult to blow at the mouth of the bottle. The pipette can be aligned with the mouth of the mouth, and the medium can be used to cover the cells close to the mouth of the bottle. After that, all the cells were aspirated and placed in a 15 ml centrifuge tube. 50 ul of the mixed cells were placed in a 1.5 ml EP tube, and 450 μl of 10% DMEM was added, which was diluted 10-fold, mixed, and 10 μl of the cells were taken. Count in the counting board. On the day of the passage, record the first day. If the next day, transfect, put 9-10×10 ⁷ /T75; if the third day is transfected, spread 3.5-4×10 ⁷ /T75. Add 15 ml of 10% DMEM medium per vial of T75. The cell density was observed on the day of transfection, and the degree of fusion reached 80-90% for transfection. px459-CVAP was transfected into RGC5 cells according to the procedure of the Lipofectamine 2000 instructions.

After transfection for 48 hours, the adherent cells were transfected by trypsinization, and the cells were collected by centrifugation. The cells were aspirated and added to 1 ml of PBS to resuspend the cells. 500 μl was placed in the original flask to continue the culture, and the remaining cells were placed in 1 A 5 ml centrifuge tube was used to extract total protein.

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

The RGC5 cells without any treatment were used as the control group. The cells obtained in the fourth example were used as experimental groups, and 100-200 μl of 5 × SDS-PAGE loading buffer was added, boiled for 5 min, and 15 μl of SDS- was loaded. PAGE protein electrophoresis. After electrophoresis, the protein was semi-dried, 10% skim milk powder was blocked for 2 h, and the blocked PVDF membrane was placed in rabbit anti-human CVAP antibody. After buffering for 3 times, the membrane was transferred to goat anti-rabbit secondary antibody. The buffer was incubated for 60 min at room temperature and rinsed 4 times with a rinse buffer. After the rinsing, the Western blotting membrane was detected by ECL development, and the results are shown in Fig. 1. It can be seen that the CVAP protein band was not detected by Western Blot in the CVAP frameshift gene mutation RGC5 cells, while the CVAP protein band appeared in the control group, indicating that the gRNA sequence for knocking out the human cell CVAP gene can realize CVAP. Knockout of genes.

Industrial applicability

Claims

A gRNA for specifically targeting a human CVAP gene in a CRISPR/Cas9 specific knockout human CVAP gene, characterized in that the target sequence of the gRNA on the human CVAP gene conforms to 5'-N(20)-NGG3' Or the sequence arrangement rule of 5'-CCN-N(20)-3', the target sequence on the human CVAP gene is unique.
The gRNA for specifically targeting a human CVAP gene in a CRISPR/Cas9 specific knockout human CVAP gene according to claim 1, wherein the targeting site is set forth in SEQ ID NO.
The gRNA for specifically targeting a human CVAP gene in a CRISPR/Cas9-specific knockout human CVAP gene according to claim 2, wherein the tumor cell is an RGC5 cell.