Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Targeted apo CIII CRISPR-Cas 9 system and application thereof

A technology of targeting and target sequence, applied in the field of sgRNA that specifically targets apoCIII gene, can solve problems such as easy generation of drug resistance, and achieve the effect of lowering blood lipid levels and reducing recovery

Active Publication Date: 2016-04-06
河北仁博科技有限公司
View PDF2 Cites 46 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, statins and benzoic acid have been used to regulate the secretion of apoCIII in the liver. Patients with hypertriglyceridemia and cardiovascular disease need to take it for a long time, which is prone to drug resistance

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Targeted apo CIII CRISPR-Cas 9 system and application thereof
  • Targeted apo CIII CRISPR-Cas 9 system and application thereof
  • Targeted apo CIII CRISPR-Cas 9 system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Design and synthesis of sgRNA specifically targeting apoCIII gene in CRISPR-Cas9 specific knockout of human apoCIII gene

[0032] 1. Design of sgRNA targeting human apoCIII gene

[0033] (1) Select the sequence of 5'-GGN(19)GG or 5'-GN(20)GG or 5'-N(21)GG on the apoCIII gene.

[0034] (2) The target site of sgRNA on the apoCIII gene is located in the exon of the gene.

[0035] (3) The targeting site of sgRNA on the apoCIII gene is located on the common exons of different splicing forms.

[0036] (4) Use BLAT in the UCSC database or BLAST in the NCBI database to determine whether the target sequence of the sgRNA is unique and reduce potential off-target sites.

[0037] According to the above method, we designed a total of 37 sgRNAs targeting the human apoCIII gene, the sequences of which are shown in the sequence table SEQ ID NO.4-40.

[0038] 2. Selection of sgRNA targeting human apoCIII gene

[0039] (1) The target sequence of the sgRNA targeting the apoCIII gene s...

Embodiment 2

[0051] Use CRISPR-Cas9 to specifically knock out the human apoCIII gene (the sgRNA sequence used to target the apoCIII gene is shown in the sequence table SEQ ID NO.12)

[0052] 1. Enzyme digestion of the linearized plasmid pL-CRISPR.EFS.GFP. After digestion, use AxyPrepPCRCleanupKit (AP-PCR-250) to purify and recover to 20-40 μl sterilized water.

[0053] 2. Connect the double-stranded sgRNA oligonucleotide obtained after denaturation and annealing that can be ligated into the vector to the linearized pL-CRISPR.EFS.GFP to obtain the pL-CRISPR.EFS.GFP-sgRNA plasmid.

[0054] 3. Transform the ligation product obtained in the above steps into competent cells and coat with Amp + Plate (50 μg / μl), and pick clones.

[0055] 4. Sequencing with universal primers to identify positive clones.

[0056] 5. Cultivate positive clones overnight on a shaker at 37°C, extract the plasmid, and obtain the pL-CRISPR.EFS.GFP-sgRNA plasmid.

[0057] 6. Cell culture and transfection.

[0058] 7....

Embodiment 3

[0060] Use CRISPR-Cas9 to specifically knock out the human apoCIII gene (the sgRNA sequence used to target the apoCIII gene is shown in the sequence table SEQIDNO.13)

[0061] 1. Enzyme digestion of the linearized plasmid pL-CRISPR.EFS.GFP. After digestion, use AxyPrepPCRCleanupKit (AP-PCR-250) to purify and recover to 20-40 μl sterilized water.

[0062] 2. Connect the double-stranded sgRNA oligonucleotide obtained after denaturation and annealing that can be ligated into the vector to the linearized pL-CRISPR.EFS.GFP to obtain the pL-CRISPR.EFS.GFP-sgRNA plasmid.

[0063] 3. Transform the ligation product obtained in the above steps into competent cells and coat with Amp + Plate (50 μg / μl), and pick clones.

[0064] 4. Positive clones were identified by sequencing with universal primers.

[0065] 5. Cultivate positive clones overnight on a shaker at 37°C, and extract the plasmid with the kit to obtain the pL-CRISPR.EFS.GFP-sgRNA plasmid.

[0066] 6. Cell culture and transf...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides a CRISPR-Cas 9 system used for preventing and / or treating hyperlipidemia and other cardiovascular diseases and a preparing method and application thereof. The CRISPR-Cas 9 system comprises an sgRNA sequence of a specific region on a specific targeting human apo CIII gene and a middle carrier comprising the sgRNA sequence, and the specific region on the apo CIII gene is selected from apo CIII gene expressed regions. The invention further provides the application of the CRISPR-Cas 9 system to preparation of drugs for preventing and / or treating hyperlipidemia and other cardiovascular diseases. By means of the prepared sgRNA of the specific targeting human apo CIII gene, the human apo CIII gene can be targeted accurately and knocked out. The preparing method is simple in step, and good in sgRNA targeting, and the knockout efficiency of the CRISPR-Cas 9 system is high.

Description

technical field [0001] The present invention relates to the field of genetic engineering, and more specifically relates to a CRISPR-Cas9 method for specifically knocking out the human apoCIII gene and a sgRNA for specifically targeting the apoCIII gene. Background technique [0002] Hyperlipidemia (Hyperlipidemia) is manifested by elevated plasma cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C) or high-density lipoprotein cholesterol (HDL-C) A state of lipid metabolism disorder, which is one of the risk factors for atherosclerosis, can involve many important organs in the whole body, causing coronary heart disease, cerebral embolism, intermittent claudication of lower limbs and other diseases. [0003] Apolipoprotein CIII (ApolipoproteinCIII, apoCIII) is a water-soluble low molecular weight (8.8kDa) protein, which is mainly synthesized by the liver and is a secreted hepatic protein. Apolipoprotein CIII is a component of high-density lipoprot...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/85A01K67/027A61K48/00A61P3/06A61P9/00
Inventor 崔健姜薇潘月
Owner 河北仁博科技有限公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com