A gene editing system, expression vector, gene editing kit and use thereof

A technology of gene editing and expression vectors, applied in the field of gene editing systems, can solve problems such as high uncertainty, susceptibility to X4-tropic HIV, treatment failure, etc., and achieve the effect of improving efficiency

Active Publication Date: 2021-12-28
广东赤萌医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] (1) In the early stage of HIV-1 infection, the virus in the body is dominated by R5 tropism, and in the late stage of infection, X4 phagocytic virus will gradually replace R5 phagocytic virus as the main virus type in the body (such as image 3 shown), thus CCR5-knockout cells are also susceptible to X4-tropic HIV at later stages of long-term treatment, leading to treatment failure
[0014] (2) Using a lentiviral vector to randomly insert the HIV membrane fusion inhibitory peptide gene into the cell genome, this scheme has high uncertainty, and the inserted gene will be silenced. Once inserted into a major disease or cancer-related gene, there is a potential Safety risks of disease and cancer
[0015] (3) This program uses shRNA to down-regulate the expression of CCR5, and cannot completely knock out CCR5 from the source
In addition, the program uses lentivirus as a carrier, which has high uncertainty and potential safety risks of pathogenicity and cancer

Method used

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  • A gene editing system, expression vector, gene editing kit and use thereof
  • A gene editing system, expression vector, gene editing kit and use thereof
  • A gene editing system, expression vector, gene editing kit and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 High-efficiency gRNA screening of CRISPR-Cas9 knockout CCR5 gene

[0054] 1. gRNA preparation

[0055] (1) Design a 20nt gRNA sequence according to the sequence of the CCR5 gene (including the exon and the intron sequence adjacent to the exon), the 3'-end of the target sequence of the gRNA on the CCR5 gene is NGG;

[0056] (2) Synthesize the sense strand and antisense strand of the target sequence with cohesive ends respectively (cacc is added to the 5'-end of the sense strand, if the first nucleotide at the 5'-end of the sense strand is not guanine G, add cacc to the sense strand Add caccG at the 5'-end of the antisense strand; add aaac at the 5'-end of the antisense strand, if the first nucleotide at the 5'-end of the sense strand is not guanine G, add C at the 3'-end of the antisense strand );

[0057] (3) The above-mentioned sense strand and antisense strand were mixed, treated at 90°C, cooled naturally to room temperature for annealing treatment, and do...

Embodiment 2

[0084] Example 2 Construction of Donor Vector for Site-directed Integration of Membrane Fusion Inhibiting Peptide DNA Sequence at CCR5 Site

[0085] 1. Construction of pDonor-EF1α-maC46-hCCR5F86 (insert fragment 5kb)

[0086] (1) Obtaining the upstream homology arm of CCR5F86 and constructing the pDonor-TALEN-EGFP-hCCR5F86-F expression vector

[0087] 1. PCR amplification of the target fragment F86-F;

[0088] Primers F86-SnaB I-L / F86-Sal I-R were used to amplify the F86-F target fragment using the HepG2 cell genome as a template.

[0089] F86-SnaB I-L (SEQ ID NO:7): 5'-gttctagtggttggctacgtagcaccatgcttgacccagtttc-3'

[0090] F86-Sal I-R (SEQ ID NO: 8): 5'-tagcttatcgataccgtcgacGTCACCACCCCAAAGGTGAC-3'

[0091] The obtained PCR product sequence contains the DNA sequence SEQ ID NO:9 of the F86 upstream homology arm;

[0092] gcaccatgcttgacccagtttcttaaaattgttgtcaaagcttcattcactccatggtgctatagagcacaagattttatttggtgagatggtgctttcatgaattcccccaacagagccaagctctccatctagtggacagggaagctagcag...

Embodiment 3

[0174] Example 3 Selection and optimization of the size of the inserted sequence for homologous recombination

[0175] 1. The Hela cell line was cultured on a 24-well plate with a plating density of 1.2×10^5 cells / well and cultured with DMEM medium (adding 10% FBS);

[0176] 2. Extract plasmids pDonor-EF1α-maC46-hCCR5F86(5kb), pDonor-EF1α-maC46-hCCR5F86-mini(3kb), pDonor-mCMV-maC46-hCCR5F86(1.6kb) and pX458 with endotoxin-free extraction kit -F86;

[0177] 3. Using Lipofectamine 3000Transfection Reagent, transform pX458-F86 and the above three homologous recombination plasmids into Hela cells respectively;

[0178] 4. From the fifth day, add G418 with a final concentration of 800μg / ml for screening, passaging once every 3-5 days, and replace each time with fresh DMEM medium containing 800μg / ml G418;

[0179] 5. On the 14th day of G418 screening, perform flow sorting, sort single cells into a 96-well plate, and put them in 37°C, 5% CO 2 and 95% relative humidity in an incuba...

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Abstract

The invention discloses a gene editing system. The gene editing system integrates the coding sequence of the membrane fusion inhibitory polypeptide into the safe harbor gene of the cell, so that the membrane fusion inhibitory peptide is expressed, but the safe harbor gene of the cell is not expressed. The invention knocks out the CCR5 gene, which is easy to operate and takes short time; the deletion of the CCR5 gene combined with the overexpression of the membrane fusion inhibitory peptide enables the cells to resist the R5 and X4 phagocytic HIV virus infection at the same time, and reduces the probability of virus mutation; membrane fusion inhibition The integration of the peptide coding sequence into the CCR5 gene locus enables the long-term stable expression of the membrane fusion inhibitor peptide.

Description

technical field [0001] The invention relates to a gene editing system, an expression vector, a gene editing kit and uses thereof. Background technique [0002] HIV (AIDS virus) is an RNA virus that can infect humans and cause human immunodeficiency. After the human body is infected with HIV, it will lead to immunodeficiency, and cause a series of opportunistic infections and tumors, that is, Acquired Immunodeficiency Syndrome (AIDS), which can lead to death in severe cases. After HIV invades the human body, it binds to the CD4 receptor on the cell surface, enters the CD4 T lymphocytes with the participation of the co-receptor CCR5 or CXCR4, and proliferates and replicates in the cells, causing a large number of CD4 T lymphocytes to lyse, and the virus is released from the cells HIV viremia is formed in the blood, which in turn triggers more CD4T lymphocyte infection and even collapses the immune system. [0003] Clinically, AIDS treatment mainly adopts combination drug, th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/90C12N9/22A61K31/7105A61K48/00A61P31/18
CPCA61K31/7105C12N9/22C12N15/907
Inventor 罗思施祝海宝陶米林梁福才黄雨亭唐忆琳刘方方
Owner 广东赤萌医疗科技有限公司
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