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

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...

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; (2) regular injections are required, such as once every two to four weeks, which increases the burden on patients ; (3) It is not easy to develop effective antibodies; (4) Only for extracellular targets; (5) Antibody drugs are expensive, etc.

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