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Method for introducing CRISPR-Cas9 system into human stem cells

A stem cell, hbb-sgRNA-cas9-t2a-gfp-sz technology, applied in the field of gene editing, can solve problems such as cell damage, and achieve the effects of fast expression time, high safety, and improved cell transfection efficiency

Inactive Publication Date: 2020-04-03
SHENZHEN CHANGENE MEDICAL TECH CO LTD
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Problems solved by technology

[0004] In view of the clinical transformation application of the CRISPR-Cas gene editing system into stem cells for gene therapy, the electroporation transfection method has better safety and higher transfection efficiency for the gene introduction of stem cells, and is the preferred transfection method among the existing transfection methods. The best choice, however, the electric field will cause direct damage to the cells, so how to improve the survival rate of stem cells after electroporation transfection is an important problem to be solved urgently

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  • Method for introducing CRISPR-Cas9 system into human stem cells
  • Method for introducing CRISPR-Cas9 system into human stem cells
  • Method for introducing CRISPR-Cas9 system into human stem cells

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Embodiment 1

[0047] The introduction of the CRISPR-Cas9 gene editing system into human umbilical cord-derived mesenchymal stem cells (human umbilicalcord-derived mesenchymal stem cells, hMSC) is used as a specific example to describe the patent of the invention in detail.

[0048] 1. Construction of HBB-sgRNA-Cas9-T2A-GFP-SZ plasmid carrying gene editing

[0049] 1.1 Transform the EF1 promoter to drive Cas9 to reduce the size of the imported gene fragment, chemically synthesize the CRISPR-Cas9 expression cassette and connect the GFP expression gene to construct the Cas9-T2A-GFP-SZ backbone plasmid; at the same time, target the human hemoglobin gene in hemophilia B (HBB, human haemoglobin beta), designed a highly targeted HBB-sgRNA, connected with the Cas-T2A-GFP-SZ plasmid to form a recombinant plasmid HBB-sgRNA-Cas9-T2A-GFP-SZ ( Figure 1-2 ).

[0050] The following is part of the DNA sequence of the HBB-sgRNA carrier (the gRNA sequence containing the HBB gene, that is, the underlined se...

Embodiment 2

[0114] The invention patent is described in detail by introducing the CRISPR-Cas9 gene editing system into human umbilical cord blood-derived hematopoietic stem cells (UB-HSC, umbilical cord blood-derived hematopoietic stem cells) as a specific example.

[0115] There are many sources of hematopoietic stem cells, such as bone marrow and peripheral blood, but compared with adult-derived hematopoietic stem cells, UB-HSC contains more early hematopoietic stem cells and has higher differentiation potential than adult-derived hematopoietic stem cells. At present, hematopoietic stem cell transplantation is used to treat various diseases, and the combination of UB-HSC and CRIPSR-Cas9 editing system has a good application prospect. However, UB-HSC is a kind of suspended stem cell, and it is a major difficulty in this technology to introduce this system efficiently into UB-HSC. The invention of this patent can make full use of the advantages of electroporation transfection to introduce...

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Abstract

The invention relates to a method for efficiently introducing a CRISPR-Cas9 gene editing system into human stem cells. The method comprises the following steps: (1) constructing and obtaining HBB-sgRNA-Cas9-T2A-GFP-SZ recombinant plasmid; (2) using a Neon system to introduce the HBB-sgRNA-Cas9-T2A-GFP-SZ recombinant plasmid into human stem cells, wherein conditions of electroporation transfectionare as follows: when the human stem cells are human mesenchymal stem cells: a pulse voltage is 1300-1700 V, a pulse duration is less than 30 ms, and a pulse number is at least once; when the human stem cells are human hematopoietic stem cells: a pulse voltage is 1450-1550 V, a pulse duration is 40 ms, and a pulse number is at least once; and (3) performing cell culture. The method uses an electroporation method to introduce the CRIPSR-Cas9 gene editing system into stem cells, and can effectively improve the survival rate of the stem cells after electroporation transfection.

Description

technical field [0001] The invention relates to gene editing technology, in particular to a method for introducing a CRISPR-Cas9 gene editing system into human stem cells. Background technique [0002] Stem cells are important raw materials in the field of modern biomedical technology research, and are also important target cells in the field of gene therapy. In gene therapy with stem cells as target cells, while transferring the gene editing system into stem cells, it is necessary to maintain their differentiation characteristics and cell activity, etc., so the challenges faced are much higher than transfection of other cells. At present, using the efficient CRISPR-Cas9 gene editing system to genetically modify stem cells is an important means of gene therapy. It can transfer exogenous genes into lineage-specific stem cells and enable them to integrate into chromosomes or serve as additional genes to maintain high blood pressure during cell division. Horizontal and durable...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/90C12N9/22
CPCC12N9/22C12N15/85C12N15/907C12N2800/107
Inventor 姜舒张芸熊斌
Owner SHENZHEN CHANGENE MEDICAL TECH CO LTD
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