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A method for site-specific editing of ccr5 gene

A gene editing technology, applied in genetic engineering, plant genetic improvement, botanical equipment and methods, etc., can solve problems such as uncontrollable, risky, fatal safety, etc.

Active Publication Date: 2022-03-04
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

[0012] Lentivirus is an integrative retrovirus. It uses lentiviral vectors to randomly integrate CCR5shRNA and TRIM5a genes into the cell genome. This is an uncontrollable and irreversible process. Once integrated into genetic sites related to physiological metabolism, disease and cancer , will cause a fatal security risk

Method used

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  • A method for site-specific editing of ccr5 gene
  • A method for site-specific editing of ccr5 gene
  • A method for site-specific editing of ccr5 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 High-efficiency gRNA screening of CRISPR-Cas9 knockout CCR5 gene

[0039] 1. gRNA preparation

[0040] (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;

[0041] (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 );

[0042] (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

[0068] Example 2 Construction of a donor vector for site-specific integration of the hTRIM5 / CypA gene at the CCR5 site

[0069] (1) Acquisition of the upstream homology arm F86-F of CCR5F86

[0070] Construction of pDonor-TALEN-EGFP-hCCR5F86-F expression vector

[0071] 1. PCR amplification of the target fragment F86-F;

[0072] 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.

[0073] F86-SnaB I-L (SEQ ID NO: 3):

[0074] 5′-gttctagtggttggctacgtagcaccatgcttgacccagtttc-3′

[0075] F86-Sal I-R (SEQ ID NO:4):

[0076] 5′-tagcttatcgataccgtcgacGTCACCACCCCAAAGGTGAC-3′

[0077] The resulting PCR product sequence contains the DNA sequence SEQ ID NO:5 of the F86 upstream homology arm F86-F:

[0078]gcaccatgcttgacccagtttcttaaaattgttgtcaaagcttcattcactccatggtgctatagagcacaagattttatttggtgagatggtgctttcatgaattcccccaacagagccaagctctccatctagtggacagggaagctagcagcaaaccttcccttcactacaaaacttcattgcttggccaaaaagagagttaattCAA...

Embodiment 3HI

[0134] The transformation of embodiment 3 HIV-1 susceptible cell lines

[0135] 1. Culture Hela-CD4 (X4 tropism-susceptible cell line) and TZM-bl (R5 tropism-susceptible cell line) cells on the wall of 24-well plate, the plating density is 1.2×10^5cells / well, use DMEM medium (Add 10% FBS) culture;

[0136] 2. Extract the plasmid pDonor-EF1α-hTRIM5 / CypA-EGFP-hCCR5F86 ( Figure 10 ) and pX458-F86;

[0137] 3. Using Lipofectamine 3000Transfection Reagent, the above two plasmids were co-transformed into Hela-CD4 and TZM-bl cell lines;

[0138] 4. From the fifth day, add G418 with a final concentration of 800μg / ml for selection, passaging once every 2-3 days, and replace each time with fresh DMEM medium containing 800μg / mL G418;

[0139] 5. After 12 days of drug screening, cells were digested, sorted by flow cytometry and inoculated into 96-well plates;

[0140] 6. Expand the culture of cells sorted into 96-well plates, select cell lines that stably and uniformly express green ...

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Abstract

The invention relates to the technical field of immunity to infectious diseases, in particular to a method for site-directed editing of the CCR5 gene. The present invention constructs a gRNA vector based on the CRISPR / Cas9 system based on the hTRIM5 / CypA gene and the human CCR5 gene; then co-transfects the gRNA vector based on the CRISPR / Cas9 system and the homologous recombination donor system into a recipient cell line, Thus, a cell line in which the CCR5 gene is site-specifically inserted into the hTRIM5 / CypA gene is obtained. Since the CCR5 site is an open site, the inserted exogenous hTRIM5 / CypA will continue to express stably without expressing the CCR5 gene, so that the cells can not only resist the infection of R5 phagocytic HIV‑1 virus, but also resist Infection by X4 phagocytic HIV virus.

Description

technical field [0001] The invention relates to the technical field of infectious disease immunity, in particular to a method for site-directed editing of CCR5 gene. Background technique [0002] HIV-1 is an RNA virus that can infect humans and cause human immunodeficiency. After the HIV-1 virus invades the human body, it binds to the CD4 receptor on the cell surface, enters the CD4 T lymphocyte with the participation of the co-receptor CCR5 or CXCR4, and proliferates and replicates in the cell, causing a large loss of CD4 T lymphocytes. Released into the blood to form HIVemia. After the human body is infected with HIV, it will lead to immunodeficiency and cause a series of opportunistic infections and tumors, with a high mortality rate. Clinically, combined drugs are mainly used to treat AIDS, that is, highly active anti-retroviral therapy (HAART), which is commonly known as "cocktail therapy". Cocktail therapy usually consists of two reverse transcriptase inhibitors and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/12C12N5/10
CPCC07K14/723C12N15/85C12N2800/80C12N2810/10
Inventor 祝海宝罗思施陶米林李恬婧陈梓珊黄雨亭
Owner 广东赤萌医疗科技有限公司
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