A kind of CRISPR-Cas9 system targeting apocIII and its application

A targeted and useful technology, applied in the field of genetic engineering, 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: 2018-10-16
河北仁博科技有限公司
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  • 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

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  • A kind of CRISPR-Cas9 system targeting apocIII and its application
  • A kind of CRISPR-Cas9 system targeting apocIII and its application
  • A kind of CRISPR-Cas9 system targeting apocIII and its application

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] Using 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 as SEQ ID NO.12)

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

[0053] 2. Connect the double-stranded sgRNA oligonucleotide obtained after denaturation and annealing 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. Shake the bacteria at 37°C overnight to culture the positive clones, extract the plasmid, and obtain the pL-CRISPR.EFS.GFP-sgRNA plasmid.

[0057] 6. Cell culture and transfection.

[0058] 7. Neomycin screenin...

Embodiment 3

[0060] Using 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 as SEQ ID NO.13)

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

[0062] 2. Connect the double-stranded sgRNA oligonucleotide obtained after denaturation and annealing 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 a kit to obtain the pL-CRISPR.EFS.GFP-sgRNA plasmid.

[0066] 6. Cell culture and transfection.

[0067] 7. Neomycin ...

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PUM

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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 (Apolipoprotein CIII, 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 lipopro...

Claims

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Application Information

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Patent Type & Authority Patents(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
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