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Method for inducing CCR5-delta32 deletion with genome editing technology CRISPR-Cas9

A genome editing and technology technology, applied in recombinant DNA technology, gene therapy, polymorphism use, etc., can solve problems such as CCR5Δ32 deletion

Pending Publication Date: 2016-05-11
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] The purpose of the present invention is to solve the problem of artificially inducing the deletion of CCR5Δ32, and to provide a method of using the genome editing technology CRISPR-Cas9 to induce the deletion of the HIV co-receptor CCR5Δ32 / Δ32, thereby providing a drug for the possible cure of AIDS

Method used

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  • Method for inducing CCR5-delta32 deletion with genome editing technology CRISPR-Cas9
  • Method for inducing CCR5-delta32 deletion with genome editing technology CRISPR-Cas9
  • Method for inducing CCR5-delta32 deletion with genome editing technology CRISPR-Cas9

Examples

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Embodiment 1

[0037] Example 1: Plasmid Reconstruction Carrying CRISPR-Cas9 System and Preparation of Lentiviral Particles Encapsulating the Reconstructed Plasmid

[0038] 1. Selection and design of gRNA

[0039] (1) According to the purpose of the experiment, we need to design a pair of gRNAs to edit and modify the CCR5 gene at the same time. However, due to the limitations of the CCR5 gene and the limitations of the PAM site, the choice of gRNA is limited. The left gRNA, such as SEQIDNo.1 , or SEQNo.3-19. The right sequence, such as SEQ ID No.2 or SEQ ID No.20-36. Or any combination of these flanking gRNAs.

[0040] (2) According to the gRNA sequence, enzyme cleavage sites were added to synthesize DNA, which were respectively connected into the lenti-CRISPR-v2 vector digested by BsmBI to obtain the reconstructed plasmid, and the correctness of the sequence was verified by sequencing. The whole experimental process, such as Figure 4 .

[0041] 2. Preparation of lentiviral particles a...

Embodiment 2

[0044] Example 2: Results of Lymphocytes Infected with Lentiviral Particles Encapsulated with the Reconstituted Lenti-CRISPR-v2 Plasmid

[0045] 1. Spread lymphocytes in a six-well plate before infection, and the total number of cells in each well is 2×10 5 , the medium used is a full medium without antibiotics. On the second day, the lentiviral particles of the lenti-CRISPR-v2 plasmid wrapped with gRNA obtained in Example 1 and the lentiviral particles of the empty lenti-CRISPR-v2 plasmid wrapped The disease particles were added to the cell culture medium, and polybrene, which can increase the efficiency of virus infection, was added at the same time. Culture medium, after 72 hours of culture, the cells were collected by centrifugation, and half of the collected cells were transferred to a new culture flask to continue culturing, and the other half was used to extract the total genomic DNA of the cells for subsequent analysis.

[0046] 2. Using the extracted cellular genomic...

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Abstract

The invention relates to a method for successfully inducing cell chemokine receptor CCR5 genes to be mutated into CCR5-delta32 deletion-type genes with a new genome editing technology CRISPR-Cas9. CCR5 is an important receptor for human immunodeficiency viruses (HIV) to invade personal host cells. CCR5-delta32 deletion means deletion of 32 basic groups occurs in a CCR5 coding region, so that the sequence after the 185th amino acid is changed, and early termination occurs. CCR5-delta32 biallelic-gene homozygous deletion has natural resistance to HIV infection, and can not be infected by HIV. By means of the method, a slow virus packaging system and the CRISPR technology are used at the same time; as the slow virus infecting host range is wide, the method can be applied to cells such as bone marrow stem cells and CD4T cells, and the CCR5-delta32 deletion-type genes hopefully become medicine for treating acquired immune deficiency syndrome or other diseases.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to a method for inducing CCR5Δ32 deletion by a new genome editing technology CRISPR-Cas9. Background technique [0002] AIDS (Acquired Immune Deficiency Syndrome, AIDS) caused by human immunodeficiency virus (Human Immunodeficiency Virus, HIV) or AIDS virus has been prevalent in the world for more than 30 years. AIDS is a severe infectious disease. HIV virus can directly attack the human immune system, leading to the deficiency of human immune function, and finally the death of patients due to various opportunistic infections and tumors. There are two main types of HIV, HIV-1 and HIV-2. The main cause of AIDS worldwide is HIV-1, and HIV-2 is limited to sporadic reports in parts of Africa and elsewhere. According to the report of the World Health Organization (WHO), by the end of 2013, there were about 35 million HIV-1 living infected people in the world, and about 30 million people had die...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867
CPCC12N15/86C12N2740/15045C12N2810/10A61K48/00C12N15/1138C12N2320/34C12N15/907C12N2740/16043C12N2310/20C12N9/22C12N15/102C12N15/11
Inventor 魏民齐春侠
Owner NANKAI UNIV
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