Application of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technique in coronary atherosclerotic heart diseases

A coronary atherosclerotic and sclerotic technology, applied in the field of medicine and biology, can solve the problems affecting the specific knockout of target genes, etc., and achieve the effects of high gene modification efficiency, large market application prospects, and simple operation techniques

Active Publication Date: 2017-11-21
广州艾迪基因科技有限责任公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented innovation improves genes that modify cells by modifying them more efficiently than previous methods while also simplifying their operations process for researchers interested in developing new treatments or therapies against cardiovascular diseases like coronary artery stenosis (CAS).

Problems solved by technology

Technicians discuss how various methods aim to reduce symptoms associated with diseased blood vessels called stenosis. These include surgery techniques, medicines, chemotherapy agents, and biological therapies involving stem cell implants. However, these approaches may result in complications ranging from reduced flow velocity and increased risk of embolism. There is thus a technical problem addressed in this patented research related to developing safer and effective ways to selectively target therapeutic targets within patients who suffer from vascular occlusion without harmful side effect on their health.

Method used

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  • Application of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technique in coronary atherosclerotic heart diseases
  • Application of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technique in coronary atherosclerotic heart diseases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1 Primer Design

[0019] 1. Construct the in vitro Cas9 expression vector, named pGEM-Cas9, which was synthesized by Inwin Biotechnology, and its sequence is a commonly used sequence in the field.

[0020] 2. Construction and preparation of vectors for transcribing the sgRNA backbone coding sequence in vitro.

[0021] The sgRNA is designed for the mmu-miR-8086 gene, and the forward oligonucleotide sequence and the reverse oligonucleotide sequence can complement each other to form a double-stranded DNA fragment with sticky ends:

[0022] F: CACCGgcacagccttggtgtctctagtcc

[0023] R: CggactagagacaccaaggctgtgcCAAA.

Embodiment 2

[0024] Embodiment 2, construct the sgRNA expression vector of mmu-miR-8086 gene

[0025] 1. Synthesis of DNA Inserts

[0026] (1) Synthesize the forward and reverse oligonucleotide sequences designed above

[0027] The oligonucleotide sequence can be specifically synthesized by a commercial company (Shanghai Sangon Co., Ltd.) based on the provided sequence.

[0028] The corresponding forward and reverse oligonucleotide sequences are annealed and annealed to form double-stranded DNA fragments with cohesive ends.

[0029] The reaction system (20μL) is as follows:

[0030] Forward oligonucleotide (10 μM): 1 μL

[0031] Reverse oligonucleotide (10 μM): 1 μL

[0032] 10×PCR buffer: 2μL

[0033] wxya 2 O: 16 μL

[0034] Put the above reaction system into the PCR machine, and carry out the reaction according to the following procedure.

[0035] Reaction procedure:

[0036] 95℃, 5min;

[0037] 80℃, 5min;

[0038] 70℃, 5min;

[0039] 59℃, 5min;

[0040] 50℃, 5min;

[0041] ...

Embodiment 3

[0063] Embodiment 3, preparation of transgenic mice

[0064] 1) The CRISPR / Cas9 injection system for mice is as follows:

[0065] pGEM-Cas9

50ng / μl

lentiCRISPR v2-sgRNA

10ng / μl

Mixed total volume

20μl

[0066] 2) injection

[0067] Use Eppendorf2xTransferManNK2 microinjector to draw 2 μl of the mixture in step 1) and inject 60 fertilized eggs. Subsequent conception.

[0068] Five days after the mice were born, the mouse nails were clipped to extract genomic DNA. The gene expression levels of miR-8086, Card3 and SHPS-1 were identified by PCR, and 30 mice without gene knockout were used as controls. Such as figure 2 As shown, compared with the control, the gene expression levels of miR-8086, Card3 and SHPS-1 in the mice were reduced to 0.2%, 11.7%, and 12.5%, respectively, compared with the control. This fully shows that miR-8086 in mice has been completely knocked out, and gene expression has been almost completely lost. Correspon...

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Abstract

The invention provides a method for specifically knocking off a microRNA-8086 gene by using CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats). The method comprises the following steps: (1) establishing a Cas9 expression vector; (2) establishing a sgRNA expression vector; and (3) mixing Cas9 and sgRNA- so as to obtain a mixed liquid, and injecting the mixed liquid into zygotes of mammals. Therefore, the purpose of fixed point knock-off of the microRNA-8086 gene is achieved.

Description

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Claims

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Application Information

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Owner 广州艾迪基因科技有限责任公司
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