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CRISPR-Cas9 system for preventing and/or treating HIV, as well as preparation method and application thereof

A cas9-pol-1, cas9-pol-2 technology, applied in the field of gene therapy drugs, can solve the problem of limited effect of inhibition

Inactive Publication Date: 2015-06-24
THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the published paper "RNA-directed Gene Editing Specifically Eradicates Latent and Prevents New HIV-1 Infection", Liu Wenhui et al. constructed a 20bp sgRNA based on two fragment sequences of the HIV gene, which can target HIV sequence 5 The HIV-1U3 site of 'LTR; due to the single site and other reasons, this system can only show that it is related to the inhibition of HIV, but the inhibitory effect is very limited

Method used

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  • CRISPR-Cas9 system for preventing and/or treating HIV, as well as preparation method and application thereof
  • CRISPR-Cas9 system for preventing and/or treating HIV, as well as preparation method and application thereof
  • CRISPR-Cas9 system for preventing and/or treating HIV, as well as preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0113] (1) Acquisition of sgRNA gene

[0114] The sgRNAs primer sequences were directly ordered from Invitrogen, and the relevant sequences are shown in Table 1.

[0115] Table 1: sgRNA primer sequences

[0116]

[0117]

[0118]

[0119]

[0120]

[0121] (2) Construct sgRNA into expression vector

[0122] a): PCR amplification of sgRNA

[0123] Reaction system: sterile ddH 2 O: 37.5 μL, 10×PCR Buffer (containing MgCl 2 ): 5 μL, 2.5 mM dNTP: 4 μL, corresponding upstream and downstream primers 1 μL each (primer concentration 50 pmol / μL), template DNA (50 ng / μL): 1 μL, PyrobestTM DNA Polymerase: 0.5 μL. After gentle shaking and mixing, PCR amplification was carried out according to the conditions shown in Table 2.

[0124] Table 2: PCR Amplification Conditions

[0125]

[0126]

[0127] b) Vector construction and identification

[0128] Use the PCR recovery kit produced by Quigen to recover the PCR amplification product according to the conventional ...

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Abstract

The invention provides a CRISPR-Cas9 system for preventing and / or treating HIV, as well as a preparation method and application thereof. The CRISPR-Cas9 system comprises sgRNA of a specific genetic locus on a specific target HIV genome, wherein the specific genetic locus on the HIV genome is selected from Gag, Env, Pol, Tat, Nef, Vif, Vpr, Vpu, 5'LTR, 3'LTR and Rev. The preparation method of the CRISPR-Cas9 system comprises the step of preparing an sgRNA fragment by means of PCR amplification. The invention further provides application of the CRISPR-Cas9 system in preparation of medicines for treating and / or preventing HIV infection. By adopting an efficient gene delivery system, the CRISPR-Cas9 system is delivered into HIV infected cells to efficiently inhibit generation of HIV, the inhibition rate is 96 percent, and the effect equivalent to that of peptide anti-HIV medicines can be achieved.

Description

technical field [0001] The present invention relates to the field of gene therapy drugs, in particular to the field of gene therapy for HIV, in particular to a CRISPR-Cas9 system for preventing and / or treating HIV and its preparation method and application. Background technique [0002] Human Immunodeficiency Virus (HIV) is a retrovirus, a lentivirus that infects the cells of the human immune system. It can cause the deficiency of the human immune system, make the human body lose resistance, and easily produce various diseases and even cancer. The disease it causes - AIDS has no effective treatment so far and is fatal. [0003] Conventional means to prevent infectious diseases are human intervention and vaccines. Human intervention (such as condoms, etc.) cannot truly eliminate HIV, but only serves as a physical coercive means to limit HIV; Exploration and development, so far no mature products have come out. Therefore, the current HIV main therapy is highly effective comb...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/55C12N15/85A61K48/00A61K38/46A61K31/7088A61P31/18
CPCA61K31/7088A61K38/46A61K48/00C12N15/11C12N15/85
Inventor 蒋兴宇刘野
Owner THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA