CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 Recombinant lentiviral vector containing gRNA sequence specifically targeting CCR5 and application thereof

A technology of recombinant lentivirus and lentiviral vector, applied in the field of genetic engineering, can solve the problem of incomplete inhibition of HIV replication and achieve high inhibition and rapid construction

Active Publication Date: 2017-11-03
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Tebas et al. reported in 2014 that they used ZFN to modify autologous CD4+ T cells to treat HIV patients, and Mock et al. reported the use of TALEN technology to knock out the CCR5 gene in 2015, which can protect cells from CCR5 Habitual HIV infection, but does not completely inhibit HIV replication
These two existing technologies are still difficult to meet the needs, so people look forward to finding a more efficient strategy to knock out the CCR5 gene

Method used

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  • CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 Recombinant lentiviral vector containing gRNA sequence specifically targeting CCR5 and application thereof
  • CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 Recombinant lentiviral vector containing gRNA sequence specifically targeting CCR5 and application thereof
  • CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 Recombinant lentiviral vector containing gRNA sequence specifically targeting CCR5 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] The features and advantages of the present invention can be further understood through the following detailed description in conjunction with the accompanying drawings. The examples provided are only illustrative of the method of the present invention and do not limit the rest of the present disclosure in any way. [Example 1] Design, synthesis and eukaryotic expression vector construction of gRNA sequence targeting CCR5 gene sequence

[0043] 1. Selection and design of gRNA targeting CCR5 gene sequence

[0044] Using the human CCR5 gene sequence as a reference sequence, use the Crispr design tool on crispr.mit.edu to find the target site with a higher score on the CCR5 gene sequence. The target site sequence is in the form of 5'-(20N)-NGG3' or 5'-CCN(20N)-3'. At the same time, the human genome sequence was compared to exclude gRNAs with high homology to the human genome sequence, and the genome sequences of CCR5 and CCR2 were compared to exclude gRNA sequences targeti...

Embodiment 2

[0062] [Example 2] The gRNA-guided CRISPR / Cas9 system of the present invention was used to verify the mutation of a specific site in the CCR5 gene in TZM-bl cells.

[0063] First, the constructed gRAN / Cas9 co-expression plasmid and the helper plasmids psPAX2 and pMD2.G were co-transfected into 293T cells. After 24 hours, the culture medium was replaced with fresh medium. After 48 hours, the culture supernatant was collected, filtered through a 0.45 μm filter membrane, and then cryopreserved. Standby at -80°C.

[0064] In order to verify whether the gRNA-guided CRISPR / Cas9 system designed in the present invention can mutate the specific site of CCR5, TZM-bl cells were infected with the constructed lentivirus, replaced with fresh medium after 12 hours of infection, and added 0.8 μg / ml of puromycin kills uninfected cells, the solution is changed every 24 hours, and the genomic DNA of the cells is extracted after 72 hours. Use the CCR5 primer CCR5-Primer to amplify the target fra...

Embodiment 3

[0095] [Example 3] Inhibition of CCR5 co-receptor expression by designed and constructed gRNA

[0096] The virus infection experiment was the same as in Example 2. After 72 hours, the expression of the CCR5 co-receptor on the surface of TZM-bl cells and Ghost-CCR5 cells was detected by flow cytometry, from figure 2 It can be seen that the CRISPR / Cas9 system can effectively inhibit the expression of CCR5 coreceptor under the guidance of our designed CCR5-gRNA7 and CCR5-gRNA8, especially in TZM-bl cells, the specific steps are as follows:

[0097] a. Discard the medium and wash the cells once with PBS;

[0098] b. Add 200 μl of 0.25% trypsin to digest the cells, and then add 300 μl of serum-containing medium to terminate the trypsinization;

[0099] c. Add PBS to 1ml, pipette the cells repeatedly until they are dispersed into single cells;

[0100] d. Transfer the cell suspension to a 1.5ml EP tube, centrifuge to pellet the cells (1,500g, 5min); wash 1-2 times with PBS, repea...

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Abstract

The invention discloses a CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 recombinant lentiviral vector containing gRNA sequence specifically targeting CCR5 and application thereof. A lentivirus of the CRISPR/Cas9 recombinant lentiviral vector containing gRNA sequence specifically targeting CCR5 gene Delta 32 region is constructed that the lentivirus can introduce cells into a CRISPR/Cas9 system specific to CCR5, double-chain breakage occurs to a specific site of CCR5 gene, a random mutation is introduced to a breakage site after repairing by means of nonhomogeneous recombinant terminal binding, and the mutation rate reaches 90% and above. As gRNA is a nonhomogeneous region of CCR5 and CCR2, detection shows that the missing efficiency of the two gRNAs is lower than 0.2%. Cells modified via the recombinant lentivirus have significantly decreased efficiency of HIV (human immunodeficiency virus) infection. The system is quick to construct, simple and low in price, and is applicable to gene therapy of acquired immune deficiency syndrome.

Description

technical field [0001] The present invention belongs to the field of genetic engineering, and more specifically relates to a CRISPR / SaCas9 method for specifically knocking out human CCR5 gene and a gRNA for specifically targeting CCR5 gene. Background technique [0002] Human immunodeficiency virus (HIV) belongs to the lentivirus genus Retroviridae, which can cause HIV virus infection and acquired immunodeficiency syndrome (AIDS). AIDS is a state of progressive failure of the body's immune system, leading to life-threatening opportunistic infections and cancers. Without treatment, the average survival time after HIV infection is only about 9 to 11 years, depending on the HIV subtype infected. Since the first clinical discovery of AIDS in 1981, more and more people have been tested for HIV infection. More than 30 years have passed, and HIV is still a global public health problem. About 34 million people are currently infected with HIV, and in 2014, 1.2 million people worldwi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N7/01A61K31/7105A61K48/00A61P31/18
CPCA61K31/7105C12N7/00C12N15/86C12N2740/15043C12N2800/80C12N2810/10
Inventor 郭德银陈宇刘杏陈述亮
Owner WUHAN UNIV
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