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SgRNAs specifically targeting to LAG-3 gene and method for specifically knocking out LAG-3 gene

A LAG-3, specific technology, applied in the field of genetic engineering, can solve the problems of only targeting extracellular targets, not easy to develop antibodies, and diversification of tumor mutations

Active Publication Date: 2018-03-02
BEIJING MICRO HELIX GENE TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the use of antibodies to target gene therapy is limited by some factors: (1) the effect of antibodies is only temporary blocking; (2) there are many kinds of inhibitory receptors, how to use multiple antibodies to block multiple inhibitory receptors at the same time There are no countermeasures for sex receptors; (3) It is not easy to develop effective antibodies; (4) Tumor mutations are diversified, and the inhibitory effect of antibodies is limited; (5) Only for extracellular targets; (6) Antibody drugs are expensive and many more

Method used

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  • SgRNAs specifically targeting to LAG-3 gene and method for specifically knocking out LAG-3 gene
  • SgRNAs specifically targeting to LAG-3 gene and method for specifically knocking out LAG-3 gene
  • SgRNAs specifically targeting to LAG-3 gene and method for specifically knocking out LAG-3 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Example 1 Design and Synthesis of Targeted sgRNA for CRISPR-Cas9 / Cpf1-specific Knockout of Human LAG-3, TIM-3 or PD-1 Gene

[0085] 1. Design of Cas-sgRNA targeting human LAG-3, TIM-3 or PD-1 gene:

[0086] (1) Select the sequence of 5'-N(21)GG or 5'-N(21)AG on the LAG-3, TIM-3 or PD-1 gene.

[0087] (2) The targeting site or cleavage site of the sgRNA on the LAG-3, TIM-3 or PD-1 gene is located in the exon of the gene, which is more likely to cause the deletion of the fragment or the frame-shift mutation, thereby reaching the gene Completely inactivate the purpose.

[0088] (3) The targeting site or cleavage site of the sgRNA on the LAG-3, TIM-3 or PD-1 gene is located on the common exons of different cleavage forms.

[0089](4) Use Blat in the UCSC database or BLAST in the NCBI database to determine whether the target sequence of the sgRNA is unique and reduce potential off-target sites.

[0090] According to the above method, the present invention designed a total...

Embodiment 2

[0123] Example 2 Construction of sgRNA gene editing plasmid

[0124] 1. Construction of Cas9 plasmid:

[0125] For the preparation method of plasmid PMH001-Cas9, please refer to the literature: Le Cong et al. Multiplex GenomeEngineering Using CRISPR / Cas Systems Science 339, 819 (2013); (DOI: 10.1126 / science.1231143), please refer to the structure diagram of plasmid PMH001-Cas9 figure 1 , the complete sequence is shown in SEQ ID NO.478.

[0126] 2. Construction of Cpf1 plasmid:

[0127] For the preparation method of plasmid PMH002-Cpf1, please refer to the literature: Zetsche Bet al. figure 2 , the complete sequence is shown in SEQ ID NO.479.

[0128] 3. Construction of single sgRNA gene editing plasmid:

[0129] (1) Linearize the PMH001-Cas9 or PMH002-Cpf1 plasmid prepared in steps 1 and 2 above.

[0130] Enzyme digestion system and conditions are as follows:

[0131] 2μg PMH001-Cas9 or PMH002-Cpf1 (400ng / μl);

[0132] 5μl 10x FastDigest Buffer / FastDigest Green Buffer;...

Embodiment 3

[0154] Example 3 Using CRISPR-Cas9 to specifically knock out the human LAG-3 or TIM-3 gene

[0155] 1. Cell culture and transfection

[0156] (1) HEK293T cells were inoculated and cultured in DMEM medium containing 10% FBS, penicillin (100 U / ml) and streptomycin (100 μg / ml).

[0157] (2) Divide into 12-well plates before transfection, and perform transfection when the density is 60%-80%.

[0158] (3) According to the operation manual of LipofectamineTM2000Transfection Reagent (Invitrogen, 11668-019), transfect 2 μg of the plasmid carrying LAG-3sgRNA or TIM-3sgRNA targeting LAG-3sgRNA or TIM-3sgRNA into each well of cells, change the medium after 6-8 hours, and add Puromycin (Merck, 540411) drug screening, harvested cells after 48 hours.

[0159] Design the experimental group and control group as shown in Table 1:

[0160] Table 1. CRISPR-Cas9 specific knockout of human LAG-3 or TIM-3 gene experimental group design

[0161]

[0162]

[0163] 2. T7EN1 enzyme digestion ...

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Abstract

Based on a CRISPR system, the invention provides sgRNAs specifically targeting to a human LAG-3 gene and a method for specifically knocking out the LAG-3 gene in human cells. The sgRNAs provided by the invention can accurately and effectively target to the human LAG-3 gene and precisely knock out the gene, and has excellent characteristics of high efficiency, short period and low cost. The methodcan be used for preparing human T cells having LAG-3 immune checkpoints knocked out, and then the human T cells are used in for tumor immunotherapy.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to a method for gene editing of cells, especially T cells, using a CRISPR-Cas9 system, specifically a method for using CRISPR-Cas9 to specifically knock out the LAG-3 gene in human T cells. Background technique [0002] Immune checkpoints are a series of immunosuppressive molecules. Under normal circumstances, immune checkpoints play an important role in maintaining self-immune tolerance and preventing the immune system from attacking its own organs in the fight against pathogenic infections. Many cancers achieve immune evasion through the expression of immune checkpoint proteins. of. By blocking immune checkpoints, restoring the body's own anti-tumor immune response, and using the body's immune function to eliminate cancer cells in the body has always been the dream of oncologists and cancer patients. [0003] Currently, the two most important and most studied immune checkpoint m...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/85C12N15/86C12N5/10C12N5/09A61K35/17A61P35/00
CPCA61K35/17C07K14/70503C07K14/70521C12N5/0636C12N15/113C12N15/85C12N15/86C12N2310/10C12N2502/30C12N2510/00C12N2810/10
Inventor 刘波任子男闫威黄园园
Owner BEIJING MICRO HELIX GENE TECH
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