CRISPR/SaCas9 system for gene therapy of AIDS

A gene and purpose technology, applied in the field of sgRNA that specifically targets the CCR5 gene, can solve the problem of no specific knockout

Inactive Publication Date: 2016-05-11
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] There is currently no CRISPR / SaCas9 system for specific knockout of the HIV-1 coreceptor CCR5

Method used

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  • CRISPR/SaCas9 system for gene therapy of AIDS
  • CRISPR/SaCas9 system for gene therapy of AIDS
  • CRISPR/SaCas9 system for gene therapy of AIDS

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] [Example 1] Design of sgRNA specifically targeting CCR5 in CRISPR / SaCas9 specific knockout of human CCR5 gene and analysis of target site sequence conservation

[0088] In the present invention, the co-receptor protein of HIV-1 virus, that is, the host protein CCR5 (NCBI gene number: NM_000579) was selected as the target, and a number of viruses in rhesus monkeys and humans were screened out according to the SaCas9 editing rules (F.AnnRanetal.2015). The target sites of all genes are conserved, and the 13 target sites are shown in attached table 1. In order to prove that the sequence of the CCR5 target site selected by the applicant is conserved in humans and rhesus monkeys, the applicant selected the 13 target sites for CCR5 The BLAST software in the NationalCenterForBiotechnologyInformationDatabase conducted a sequence conservation analysis of the target sites, and the results showed that 10 target sites were conserved between human and rhesus monkeys, and 3 had one bas...

Embodiment 2

[0092] [Example 2] Construction of recombinant lenti-CRISPR and AAV-CRISPR and identification of gene editing effects

[0093] The lentiviral vector selected in the present invention is lentiCRISPR / SaCas9, which is constructed by our laboratory. Specifically, the SaCas9 gene on the Addgene AAV-CRISPR-SaCas9 (also called PX601) vector is amplified by PCR and transferred to lentiCRISPR-V2 On the carrier, replace the original SpCas9. The crRNA corresponding to SaCas9 on the PX601 vector was amplified by overlapping PCR and then transferred to the lentiCRISPR-V2 vector to replace the crRNA corresponding to the original SpCas9.

[0094] The vector contains a human U6 promoter to mainly regulate the expression of sgRNA, and also contains an EFs promoter to regulate the expression of SaCas9. The modified adeno-associated virus vector is pAAV-CRISPR, which also contains a U6 promoter that controls the expression of sgRNA, but contains a TBG promoter to regulate the expression of SaCa...

Embodiment 3

[0123] [Example 3] Packaging of CRISPR / SaCas9 lentiviral system and adeno-associated virus system

[0124] In order to achieve better excision efficiency of CRISPR / SaCas9 recombinant lentivirus and adeno-associated virus vectors, the applicant packaged CRISPR / SaCas9 recombinant lentivirus and adeno-associated virus vectors into lentivirus and adeno-associated virus vectors in HEK-293T cells .

[0125] The packaging system of the lentivirus system is as follows:

[0126] psPAX2 lentiviral helper packaging plasmid (purchased from Addgene)

[0127] pMD2.G lentiviral helper envelope plasmid (purchased from Addgene)

[0128] lentiCRISPR vectors for different targets

[0129] The specific process is as follows:

[0130] 1. Spread HEK-293T cells (purchased from CCTCC) 24 hours before transfection in a cell culture dish with a diameter of 10 cm;

[0131] II. When the cell density reached 80-90%, the above-mentioned plasmids were co-transfected into the cells with the standard Lip...

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Abstract

The invention belongs to the field of gene engineering, and particularly relates to a method for specifically knocking out CCR5 genes of human bodies through CRISPR/SaCas9 and sgRNA used for specifically targeting CCR5 genes. The invention provides the method for specifically knocking out the CCR5 genes of the human bodies through CRISPR/SaCas9 and sgRNA used for specifically targeting the CCR5 genes and relates to a CRISPR/SaCas9 recombinant lentivirus and adeno-associated virus vector system and application. CRISPR/SaCas9 recombinant lentivirus and adeno-associated virus vectors can express SaCas9-RNA aiming at the CCR5 target spot of one same gene of the human bodies and rhesus monkeys, therefore, the application range is wider, and the efficiency is higher. The preparation method is easy to construct, the targeting efficiency is high, and a novel technology is provided for the gene therapy of HIV.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and more specifically relates to a CRISPR / SaCas9 method for specifically knocking out the human CCR5 gene and an sgRNA for specifically targeting the CCR5 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, and acquired immunodeficiency syndrome (AIDS) caused by its infection is one of the main diseases threatening human health worldwide one. Human Immunodeficiency Virus is also commonly known as "HIV". As of the end of 2014, there were as many as 36.9 million people living with HIV. Twenty-five years since the first case of AIDS caused by HIV was discovered in 1981, although the clinical treatment of AIDS has made great progress, there is still no effective cure to break through this scientific problem. [0003] Virus-specific CD4+ T cells are an importan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/867C12N15/864A61K48/00A61P31/18
CPCC12N15/113C07K14/47C12N15/86C12N2740/15043C12N2740/15045C12N2750/14345C12N2800/107C12N2800/80
Inventor 郭德银陈述亮肖巧巧刘哲鹏
Owner WUHAN UNIV
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