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CRISPR/SaCas9 based specific human CXCR4 gene knockout method

A specific and genetic technology, applied in the field of genetic engineering, can solve the problems of large SpCas9 gene and insufficient targeting accuracy of SpCas9, and achieve high specificity

Inactive Publication Date: 2017-10-10
上海捷易生物科技有限公司
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Problems solved by technology

[0011] Using SpCas9 to target CXCR4 for AIDS treatment has the following problems: 1) SpCas9 gene is too large to use AAV vectors for gene therapy; 2) SpCas9 has insufficient targeting accuracy

Method used

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  • CRISPR/SaCas9 based specific human CXCR4 gene knockout method
  • CRISPR/SaCas9 based specific human CXCR4 gene knockout method
  • CRISPR/SaCas9 based specific human CXCR4 gene knockout method

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Embodiment Construction

[0026] In order to have a more specific understanding of the technical content, features and effects of the present invention, the technical solutions of the present invention will be further described in detail in conjunction with the accompanying drawings and specific embodiments.

[0027] 1. Design of sgRNA specifically targeting human CXCR4 gene

[0028] Taking the co-receptor protein of HIV-1, the host protein CXCR4 (NCBI gene number: NM_001008540.2) as the target, according to the SaCas9 editing rules (F.Ann Ran et al.2015), the CXCR4 gene was selected to meet the 5'-( 20N) Sequence of NNGRRT-3'.

[0029] The targeting site of the sgRNA on the CXCR4 gene is located in the coding region of the common exon in different splicing forms (the targeting site is located in the coding region of the exon of the gene, which is more likely to cause frameshift mutation of the gene, reaching for the purpose of complete gene inactivation), avoiding regions of CpG islands.

[0030] Us...

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Abstract

The invention discloses sgRNA for specific targeting of a human CXCR4 gene, a sgRNA-containing staphylococcus aureus CRISPR / SaCas9 system and a method for specific human CXCR4 gene knockout by the method. A target sequence of sgRNA is shown as any one of SEQ ID NO:1-4. sgRNA for specific targeting of the human CXCR4 gene and AAV-CRISPR / SaCas9 plasmids are connected to form a carrier and packaged into AAV infection cells, simplicity, convenience, high efficiency and specificity in CXCR4 gene knockout are realized, and accordingly the problem of limitation in adoption of SpCas9 targeted CXCR4 for treatment of acquired immune deficiency syndrome is effectively solved.

Description

technical field [0001] The present invention relates to the field of genetic engineering, in particular to a method for specifically knocking out the human CXCR4 gene based on the Staphylococcus aureus CRISPR / Cas9 (CRISPR / SaCas9) system, and an sgRNA for specifically targeting the human CXCR4 gene. Background technique [0002] Human immunodeficiency virus (human immunodeficiency virus, HIV) belongs to the human lentivirus group in the genus Lentivirus of the family Retroviridae. It is a lentivirus that infects cells of the human immune system. Acquired immunodeficiency syndrome (AIDS), also known as AIDS, caused by its infection is a very harmful infectious disease. Since its discovery more than 30 years ago, AIDS has caused more than 20 million deaths, and has always been a huge public health problem, affecting the lives of 35.3 million people around the world. [0003] HIV can be divided into two subtypes, HIV-1 and HIV-2. HIV-1 is highly pathogenic and is the main patho...

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Application Information

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IPC IPC(8): C12N15/113C12N9/22C12N15/90C12N15/864
CPCC12N9/22C12N15/113C12N15/86C12N15/902C12N2310/10C12N2750/14143
Inventor 杨通刘佩欧恩智陈珺朱智
Owner 上海捷易生物科技有限公司
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