sgRNA (singleguide Ribonucleic Acid), lentiviral vector constructed by the same and application thereof

A lentiviral vector and recombinant lentivirus technology, applied in the field of lentiviral vector and sgRNA, can solve the problems of affecting gene modification, limited technical means, complicated application, etc., and achieve the effects of low cost, high infection efficiency and convenient operation.

Inactive Publication Date: 2017-06-06
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In previous studies, zinc finger nuclease (ZFN) or transcription activator-like effector nuclease (TALEN) was used to specifically modify the embryonic cells of rhesus monkeys to obtain transgenic monkeys, but for differentiated peripheral primary The genetic modification of cells, the technical means that can be used are limited
Using adenovirus or adeno-associated virus as a carrier to deliver ZFN or TALEN can moderately modify peripheral blood cells, but the application of these two technologies is more complicated, and adenovirus itself is immunogenic to rhesus monkeys, which may affect gene modification Effect

Method used

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  • sgRNA (singleguide Ribonucleic Acid), lentiviral vector constructed by the same and application thereof
  • sgRNA (singleguide Ribonucleic Acid), lentiviral vector constructed by the same and application thereof
  • sgRNA (singleguide Ribonucleic Acid), lentiviral vector constructed by the same and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Construction of the SIV-CRISPR / Cas9 lentiviral expression vector pCL20-CAS-sgRNA targeting human / monkey CXCR4 / CCR5

[0043] (1) Search the CXCR4 gene and CCR5 gene of human and rhesus monkey from the GenBank database, and find the homologous sequences of the protein coding regions of the two through sequence comparison. Select the homologous sequence near the 5' end, and use the online tool (http: / / crispr.mit.edu / ) to design possible sgRNAs. This online tool can combine the possible off-target probability according to the base distribution of the target sequence to obtain a comprehensive score. The higher the sgRNA score, the higher the possible knock-out efficiency and the lower the off-target efficiency. Based on this, in this patent, 7 sgRNAs ranked top and close to the 5' end of the coding region were selected for CXCR4; 8 sgRNAs ranked top and close to the 5' end of the coding region were selected for CCR5, such as Figure 1(A)-Figure 1(B) shown.

[0...

Embodiment 2

[0055] Example 2: Large-scale extraction of lentiviral expression vectors and packaging vectors

[0056] (1) Take a small amount of pCL20-CAS-sgRNA, pCAG4-RTR-SIV, pCAG-SIVgprre, and pCAG-VSVG plasmids to transform stbl3 Escherichia coli competent, and pick a single clone to inoculate in 1ml of ampicillin (Amp) containing ampicillin (Amp) LB culture solution, after shaking the bacteria for 8 hours, take 500μl of the bacteria solution into a culture bottle containing 500ml of LB / Amp culture solution, and incubate on a shaker at 37°C for 12-16 hours;

[0057] (2) Centrifuge at 5000*g for 5 minutes, collect the bacteria, discard the culture medium, turn it upside down on absorbent paper and pat lightly to absorb the residual liquid. The collected bacteria were purified using a plasmid extraction kit (Guangzhou Meiji Biotechnology Co., Ltd.).

[0058] (3) Add 20ml BufferP1 / RNase A to the cells, vortex to resuspend the bacteria (make sure the bacteria are completely resuspended, a...

Embodiment 3

[0079] Example 3: Packaging, concentration and titration of lentivirus.

[0080] 3.1 Packaging of lentivirus

[0081] (1) Cell pre-plating: 24 hours before transfection, take the overgrown 293T cells for pre-plating, and it is advisable to reach 70% confluence the next day (pre-plate 5 million in a 75mm culture dish);

[0082] (2) The next day: transfection;

[0083] (a) Observe the growth of the cells on the pre-plate. It is advisable that the confluence of the cells is about 90%, absorb the medium, and add fresh preheated complete medium (15mL in a 75mm culture dish);

[0084] (b) Before transfection, place PEI, plasmid, Opti-MEM I Reduced Serum Medium, etc. required for transfection at room temperature to equilibrate;

[0085] (c) Prepare a mixture of plasmids. A total of 10 dishes of 293T cells were packaged this time, and 500 μL of the cell mixture was prepared for each dish, so the total volume of the mixture to be prepared was 5 mL. First add three kinds of plasmids a...

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Abstract

The invention relates to the field of gene therapy, in particular relates to sgRNA (singleguide Ribonucleic Acid), as well as a lentiviral vector constructed by the same and application thereof and specifically relates to sgRNA with SIVmac1A11 lentivirus as a framework to express SpCas9 protein and gene specificity. The sgRNA is applied to treating human and simian AIDS (Acquired Immune Deficiency Syndrome). A nucleotide sequence of the sgRNA is shown as SEQ ID NO.1 to 2. According to the sgRNA disclosed by the invention, a current most efficient CRISPR / Cas9 gene editing tool is utilized, a designed CXCR4 / CCR5 gene sgRNA locus has gene knockout activity superior to other loci reported by existing research, and the sgRNA is applied to gene therapy of SIV infected rhesus monkeys for the first time. Compared with ZFN and TALEN, the sgRNA has the advantages of being convenient to operate, low in cost and the like.

Description

technical field [0001] The present invention relates to the field of gene therapy, in particular to a sgRNA and its constructed lentiviral vector and application, specifically SIVmac 1A11 Lentivirus is used as the backbone to express SpCas9 protein and gene-specific sgRNA for the treatment of human and monkey AIDS. Background technique [0002] Clustered regularly interspaced short palindromic repeats and its associated Cas9 protein system (CRISPR / Cas9) are a natural defense mechanism used by bacteria and archaea to resist foreign virus infection. After the exogenous DNA invades bacteria / archaea, it will be recognized by the RNA guide sequence (guideRNA) complementary to the specific region of the exogenous DNA in the cell, and guide Cas9 nuclease to reach the recognition site to digest the target sequence, thereby degrading the exogenous DNA. source DNA. The specific working principle of this system is as follows: crRNA (CRISPR-derived RNA) combines with tracrRNA (trans-a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/867C12N15/66C12N7/01A61K48/00A61P31/18
CPCA61K48/0025A61K48/005C07K14/705C12N7/00C12N15/113C12N15/86C12N2310/10C12N2740/15043C12N2800/80
Inventor 姚永超陈小平余松林肖宏奎李姣姣秦莉赵思婷
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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