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CRISPR-Cas9 system for knocking out gene GRIN2D and its application

A gene and DNA sequence technology, applied in the field of cancer precision medicine, can solve the problems of no knockout and no research on the effect of gastric cancer cell function, and achieve simple operation, inhibition of proliferation, invasion and migration, and high knockout efficiency Effect

Active Publication Date: 2018-09-04
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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  • CRISPR-Cas9 system for knocking out gene GRIN2D and its application
  • CRISPR-Cas9 system for knocking out gene GRIN2D and its application
  • CRISPR-Cas9 system for knocking out gene GRIN2D and its application

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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 gene GRIN2D and its application. The CRISPR-Cas9 system comprises Cas9 and gRNA specifically targeting to the gene GRIN2D. The invention also discloses application of the CRISPR-Cas9 system to preparation of drugs used for treating cancer and application of the gene GRIN2D to diagnostic kits and medicines used for precise treatmentof stomach cancer and other tumors. In virtue of a clinical sample tissue chip of stomach cancer, the gene GRIN2D is proved to be increased in expression in stomach cancer tissue and be related to prognosis according to immunohistochemical results. The CRISPR-Cas9 system provided by the invention can highly efficiently knock out the highly-expressed gene GRIN2D in stomach cancer and inhibit the proliferation and migration / invasion of gastric cancer cells, and is simple to operate and high in knockout efficiency. The CRISPR-Cas9 system is expected to be applied to diagnosis and treatment of stomach cancer and other tumors with over-expressed GRIN2D. The CRISPR-Cas9 system is applicable to a plurality of 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 Applications(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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