A CRISPR-Cas9 system for knocking out the Grin2D gene and its application

A gene and gene knockout technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of no knockout, no research on the effect of gastric cancer cell function, etc., to achieve simple operation, inhibition of proliferation and invasion and migration. , the effect of high knockout efficiency

Active Publication Date: 2021-10-22
AFFILIATED HOSPITAL OF NANTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

According to the existing literature, the expression of GRIN2D in gastric cancer and its relationship with clinical features and prognosis, and the effect of GRIN2D on the function of gastric cancer cells have not been studied
At the same time, there is no report on the CRISPR-Cas9 system for knocking out the GRIN2D gene

Method used

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  • A CRISPR-Cas9 system for knocking out the Grin2D gene and its application
  • A CRISPR-Cas9 system for knocking out the Grin2D gene and its application
  • A CRISPR-Cas9 system for knocking out the Grin2D gene and its application

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Experimental program
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Embodiment 1

[0033] 1.1 Target site design and synthesis

[0034] (1) Designing Crispr / cas9 knockout target sites

[0035] First, a pair of oligo DNA of about 20 bp needs to be designed in the target DNA region, and designed through an online tool: CRISPR Design of the Massachusetts Institute of Technology: http: / / crispr.mit.edu / .

[0036] Select the CDS region of all transcripts of the GRIN2D gene, and find out the exons in the common CDS region for target site design. The GC content of the first exon is too high, and the target site is designed on the second exon information:

[0037] GRIN2D-gRNA1: 5'-CTGACTGACGGTAGTCTGGTGGG-3'

[0038] GRIN2D-gRNA2: 5'-ACGAGTGGTCACGGCTACAAAGG-3'

[0039] GRIN2D-gRNA3: 5'-GACCTGAAGCTGTTGCTCGGTGG-3'

[0040] (2) Primer Add Adapter

[0041] Primer synthesis needs to add extra bases to the head of the target sequence, add CACC to the forward primer, and add AAAC to the reverse primer. Special attention should be paid to the fact that the first base of ...

Embodiment 2

[0093] 2.1 Human gastric cancer tissue samples

[0094] 454 cases of gastric cancer tissue specimens were collected from surgical resection specimens of gastric cancer in the Affiliated Hospital of Nantong University from 2002 to 2009, all of which had complete clinicopathological data and follow-up data. Surgical specimens were fixed with 40g / L neutral formaldehyde, embedded in paraffin, serially sectioned with a thickness of 3 μm, and subjected to HE and immunohistochemical staining respectively; all cases were not treated with radiotherapy and chemotherapy before operation. Among the patients, there were 106 males and 384 females. The TNM staging of gastric cancer adopts the AJCC standard (2002). The tissue type adopts the Lauren classification, which is divided into intestinal type, diffuse type and mixed type. In addition, 68 cases of adjacent non-tumor gastric mucosa were randomly selected from 454 cases of gastric cancer as controls. Another 31 patients with chronic ...

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Abstract

The invention discloses a CRISPR-Cas9 system for knocking out the GRIN2D gene and its application. The CRISPR-Cas9 system includes Cas9 and a gRNA specifically targeting the GRIN2D gene. The invention also discloses its application in the preparation of drugs for treating cancer and the application of GRIN2D gene in the preparation of diagnostic kits and drugs for precision medicine of gastric cancer and other tumors; it is confirmed by immunohistochemistry through tissue chips of clinical samples of gastric cancer, The expression of GRIN2D is increased in gastric cancer and is related to prognosis; the CRISPR-Cas9 system of the present invention can efficiently knock out the GRIN2D gene highly expressed in gastric cancer, and inhibit the proliferation, invasion and migration of gastric cancer cells. The system is simple to operate and has high knockout efficiency. It is expected to be applied in the diagnosis and treatment of gastric cancer and other tumors that overexpress GRIN2D. The system of the present invention is applicable to various tumors with abnormal expression of GRIN2D.

Description

technical field [0001] The present invention relates to the technical field of precision medicine for cancer, and specifically relates to a knockout of the GRIN2D gene (derived from the NCBI GENE database, ID: 2906) and the use of the GRIN2D gene as a diagnosis and treatment target for gastric cancer and other tumors. Background technique [0002] According to the statistics of GLOBOCAN in 2012, globally, there are approximately 952,000 new cases of gastric cancer each year, and approximately 723,000 deaths due to gastric cancer each year. The global incidence of gastric cancer ranks fifth among malignant tumors. About 70% of the patients are in developing countries, and 1 / 2 of the patients are concentrated in East Asia. South Korea is the country with the highest incidence of gastric cancer in the world, followed by Mongolia, Japan and China; the incidence of gastric cancer in my country ranks third among malignant tumors. The death rate ranks third, with about 400,000 new ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/90C12N15/12A61K31/7088A61P35/00C12Q1/6886
CPCA61P35/00A61K31/7088C12N15/907C12Q1/6886C07K14/47C12N2800/107C12N2810/10C12Q2600/158C12Q2600/156A61K31/7105A61K31/711A61K2300/00
Inventor 黄剑飞李洁莹
Owner AFFILIATED HOSPITAL OF NANTONG UNIV
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