Method for improving efficiency of homologous repair in CRISPR/CAS9 gene editing

A technology for gene editing and repair efficiency, applied in the field of gene editing, can solve the problems of low success rate of gene editing individuals, low proportion of homozygous homozygotes for homologous repair, difficulty in positive cell selection, etc., and achieves simple operation, low cost and obvious effect Effect

Inactive Publication Date: 2018-08-24
CHINA AGRI UNIV
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Problems solved by technology

[0004] The purpose of the present invention is to provide a new method for improving the efficiency of homologous repair in CRISPR / CAS9 gene editing, to solve the problem of low homologous repair efficiency in cells (including single-cell fertilized eggs), low homozygous ratio of homologous repair, and positive Difficulty in cell screening and low success rate of individual gene editing

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  • Method for improving efficiency of homologous repair in CRISPR/CAS9 gene editing
  • Method for improving efficiency of homologous repair in CRISPR/CAS9 gene editing

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Embodiment 1

[0026] Example 1 Method for Improving Homologous Repair Efficiency in CRISPR / CAS9 Gene Editing

[0027] This example takes the repair of myostatin (MSTN) gene as an example, and provides a method for improving the efficiency of homologous repair in CRISPR / CAS9 gene editing.

[0028] 1. MTR calculation method

[0029] 10 cultured in vitro for in vitro transfection 6 cell as an example, the MSTN allele content is 0.34×10 - 5 pMol, if the MTR value is 5 × 10 5 , then the repair template DNA (homology arm construct) is 1.7 pMol. If the repair template DNA contains carrier redundant sequences, only calculate the molecular weight of the DNA including the homology arms, and then calculate the amount (pg or pM) of the repair template DNA that needs to be added.

[0030] 2. In vitro gene editing and homologous repair of fetal fibroblasts

[0031] 2.1 Culture of fetal fibroblasts in vitro

[0032] Fetuses of Laoshan dairy goat, Landrace pig, Jinnan cattle, etc. were collected for...

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Abstract

The invention provides a novel method for improving the efficiency of homologous repair in CRISPR / CAS9 gene editing. According to the novel method, gene editing is carried out by utilizing a CRISPR / CAS9 system, the MTR value is controlled within 1x10<5>-5x10<5>, so that the homologous repair efficiency is improved. The MTR value is the ratio of the mole number of the exogenous gene DNA to be typedin to the mole number of the genes to be targeted. For the method, according to the self homologous repair mechanism and the cell cycle feature of cells, the use amount of the repair template DNA isincreased in the gene editing process, so that a rich template is provided for the accurate gene repair, further, the ratio of the non-homologous end joining is reduced, and therefore, the balance ofthe repair tends to the homologous repair. With the method, the gene repair efficiency of the in-vitro culture of somatic cells can be promoted to 90% or above, which is higher than that of the international existing method by 30%, and thus the method has the broad application prospect.

Description

technical field [0001] The invention relates to gene editing technology, in particular to a method for improving homologous repair efficiency in CRISPR / CAS9 gene editing. Background technique [0002] Repairing genetic defects at the cellular and living levels has become the primary means and method for cell therapy of diseases, development of new drugs, and improvement of animal phenotypes. In the cell therapy of any animal, the living cells of the animal are collected, isolated and cultured in vitro, the defective gene is corrected by gene editing technology, and the quality is controlled and then returned to the animal to achieve the purpose of treating certain diseases. Cell therapy has developed into the main development direction of modern medicine. Animal gene editing is mainly used to obtain high value-added products such as improved production performance, no immune rejection genes (realization of humanization), and large-scale production of biologically active sub...

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Application Information

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IPC IPC(8): C12N15/90
CPCC12N9/22C12N15/907
Inventor 李岩于坤连正兴邓守龙连玲
Owner CHINA AGRI UNIV
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