Method for specifically knocking out human PCSK9 gene by virtue of CRISPR-Cas9 and sgRNA for specifically targeting PCSK9 gene

A pcg-u6-hpcsk9sg, specific technology, applied in the field of genetic engineering, can solve the problems of increasing patient burden, expensive antibody drugs, and difficulty in developing antibodies, etc., to achieve effective and permanent effects

Inactive Publication Date: 2016-08-24
沈志荣
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the use of PCSK9 antibodies to treat hyperlipidemia also has certain limitations
Because the use of antibodies for target gene therapy is still limited by some factors: (1) the effect of antibodies is only a temporary blocking effect;

Method used

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  • Method for specifically knocking out human PCSK9 gene by virtue of CRISPR-Cas9 and sgRNA for specifically targeting PCSK9 gene
  • Method for specifically knocking out human PCSK9 gene by virtue of CRISPR-Cas9 and sgRNA for specifically targeting PCSK9 gene
  • Method for specifically knocking out human PCSK9 gene by virtue of CRISPR-Cas9 and sgRNA for specifically targeting PCSK9 gene

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Experimental program
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Embodiment 2

[0083] Example 2 Using CRISPR_Cas9 to specifically knock out the human PCSK9 gene (the sgRNA used to target the PCSK9 gene is shown in the sequence table SEQ ID NO..46, 49, 51, 52, 54 or 70, taking 46 as an example)

[0084] 1. The pCG-U6-sgRNA plasmid whose linearization sequence is shown in the sequence table SEQ ID NO..458.

[0085] Enzyme digestion system and conditions are as follows:

[0086] 2 μg pCG-U6-sgRNA (400ng / μL);

[0087] 1μL CutSmart Buffer;

[0088] 1 μL BfuAI (NEB, R0701L);

[0089] Replenish water to 50 μL, incubate at 37°C for 3-4 hours, shake and centrifuge at intervals to prevent droplets from evaporating

[0090] onto the tube cap.

[0091] After digestion, use AxyPrep PCR Clean up Kit (AP-PCR-250) to purify and recover to 20-40 μL sterilized water.

[0092] 2. The double-stranded sgRNA oligonucleotide (its Forward oligo and Reverse oligo sequences are respectively shown in the sequence table SEQ ID NO..466 and 467) that can be connected to the U6 e...

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Abstract

The invention relates to the technical field of genetic engineering, in particular to a method for specifically knocking out a human PCSK9 gene by virtue of CRISPR-Cas9 and sgRNA for specifically targeting the PCSK9 gene; through high-throughput cloud computing and designing, a group of sgRNA for specifically knocking out the specifically targeted PCSK9 gene in the human PCSK9 gene by virtue of the CRISPR-Cas9 is synthesized; meanwhile, each sgRNA is linked to a linear pCG-U6-SgRNA plasmid, so that a carrier is obtained, and a pair of forward and reverse sgRNA oligonucleotide carriers, together with the pCG-NLS-FLAG-Cas9 plasmid, are used for successfully transfecting cells, so that the PCSK9 gene is knocked out; the method has the advantage that the large-scale production of the sgRNA can be achieved just by synthesizing a few of polynucleotide segments; and the preparation method is simple and easy to implement, good in sgRNA targeting properties and high in knocking-out efficiency on the CRISPR-Cas9 system.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a CRISPR-Cas9 method for specifically knocking out the human PCSK9 gene and an sgRNA for specifically targeting the PCSK9 gene. Background technique [0002] The clustered regularly interspaced short palindromic repeat system (clustered regularly interspaced short palindromic repeat; CRISPR-associated, CRISPR-Cas9) is a complex with endonuclease activity, which recognizes a specific DNA sequence and cuts at a specific site to form a double Strand DNA breaks (Double-strandbreaks, DSB), in the absence of template conditions, non-homologous end joining (Non-homologous endjoining, NHEJ), resulting in frameshift mutation (frameshift mutation), leading to gene knockout (such as figure 1 ). [0003] This technology has attracted widespread attention because it can quickly, easily and efficiently target any gene in the genome and became popular in 2012. Due to its advantage...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/85C12N15/57
Inventor 沈志荣王凤超
Owner 沈志荣
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