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Method for improving fixed-point integration of genes into cells based on electroporation technology and CRISPR-Cas9 technology

A fixed-point integration and gene technology, which is applied in chemical instruments and methods, botanical equipment and methods, biochemical equipment and methods, etc. Effect

Inactive Publication Date: 2020-11-17
杭州普科亭生物医药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The CRISPR-Cas9 system has made great achievements in gene knockout, but the proportion of site-specific insertion of genes is still very low, whether it is a stable cell line or primary cells

Method used

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  • Method for improving fixed-point integration of genes into cells based on electroporation technology and CRISPR-Cas9 technology
  • Method for improving fixed-point integration of genes into cells based on electroporation technology and CRISPR-Cas9 technology
  • Method for improving fixed-point integration of genes into cells based on electroporation technology and CRISPR-Cas9 technology

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Experimental program
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Embodiment 1

[0037] 1. Synthesis of CAR gene and homology arm gene

[0038] Taking the target CD19-CAR currently used in clinical treatment of leukemia as an example, it is constructed in an adeno-associated virus (AAV) vector. The amino acid sequence of the CAR gene is shown in SEQ ID NO: 1, and the structural diagram is shown in figure 1 As shown, it mainly includes the ScFv that recognizes the specific tumor antigen CD19, the extracellular CD28 transmembrane region, the co-stimulatory signal CD28, and the activation signal CD3zeta. The gene sequence of the left homology arm is shown in SEQ ID NO: 2, and the gene sequence of the right homology arm is shown in SEQ ID NO: 3. The CAR gene and homologous arm gene fragments were synthesized by PCR, the forward primer was synthesized by PCR as shown in SEQ ID NO:4, and the reverse primer was shown as SEQ ID NO:5.

[0039] 2. The CAR gene and the homology arm gene are constructed on the adeno-associated virus vector and the virus is produced ...

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Abstract

The present invention discloses a method for improving fixed-point integration of genes into cells based on an electroporation technology and a CRISPR-Cas9 technology. The method comprises the following steps: designing and synthesizing a target gene aiming at a specific tumor antigen and synthesizing homologous arms on two sides of the target gene; constructing the synthesized target gene and homologous arms on adeno-associated virus vectors to produce viruses; synthesizing guide RNA for recognizing an insertion site, and expressing and purifying Cas9 protein; and after combining the guide RNA and Cas9 protein in vitro, mixing the combined guide RNA and Cas9 protein with cells, carrying out electroporation, adding the adeno-associated viruses with the target gene, and detecting expressionof the target gene and the functions of the cells. The method for improving the fixed-point integration of genes into cells is based on the electroporation technology and the CRISPR-Cas9 technology,the adeno-associated viruses serve as the vectors for gene insertion, and an efficient fixed-point insertion effect can be achieved.

Description

technical field [0001] The invention relates to site-specific insertion of genes, specifically a method for improving site-specific integration of genes into cells by using electroporation technology combined with CRISPR-Cas9 technology, and is used for the treatment of leukemia. Background technique [0002] The current method of integrating genes into cells is mainly through traditional lentiviruses and retroviruses, but the insertion of this method is random insertion. If foreign genes can be inserted into cells at a fixed point to achieve the purpose of treating diseases, it will be possible in the future. play a greater role clinically. The CRISPR-Cas9 system has made great achievements in gene knockout, but the proportion of gene-directed insertion is still very low, whether it is a stable cell line or a primary cell. [0003] If the target gene can be inserted into the cell by modifying the CRISPR-Cas9 technology, so as to realize the target gene integration on the c...

Claims

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Application Information

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IPC IPC(8): C12N15/864C12N5/10
CPCC07K14/47C12N15/86C12N2750/14143
Inventor 王甜
Owner 杭州普科亭生物医药有限公司