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Method for improving efficiency of genome site-directed modification by using small molecular compounds

A technology of small molecule compounds and genome targeting, applied in the field of genetic engineering, can solve the problem of low efficiency of genome targeting modification

Active Publication Date: 2019-11-26
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] In order to overcome the defect of low efficiency of genome-specific modification, the present disclosure provides a method for improving the efficiency of genome-specific modification to solve this problem

Method used

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  • Method for improving efficiency of genome site-directed modification by using small molecular compounds
  • Method for improving efficiency of genome site-directed modification by using small molecular compounds
  • Method for improving efficiency of genome site-directed modification by using small molecular compounds

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Example 1: Screening of small molecular compounds that improve the efficiency of genome-specific modification

[0032] 1 vector construction

[0033] 1.1 Gene synthesis of green fluorescent protein (GFP) reporter carrier

[0034] The GAPDH-GFP reporter gene was synthesized by Nanjing GenScript Biotechnology Co., Ltd., as follows: figure 1 As shown in A, the GAPDH-GFP reporter carrier model diagram: After CRISPR / Cas9 cuts the GAPDH gene to generate DSB, the cells use the donor GAPDH-T2A-GFP plasmid or the donor GAPDH-T2A-GFPCR product template to undergo HDR repair or NHEJ repair, if If homologous repair occurs, the tandem green fluorescent protein gene can be expressed, if no homologous repair occurs, there will be no fluorescent protein; figure 1 Shown in B, ssODN-GFP reporter carrier model diagram: a CRISPR / Cas9-ROSA26 targeting sequence SEQ ID No: 1: 5'-GTGAGAGTTATCTGACCGTAAGG-3' is inserted in the middle of the reporter carrier EGFP sequence. When CRISPR / Cas9 cuts...

Embodiment 2

[0053] Example 2: Using Small Molecular Compounds to Improve the Efficiency of Targeted Genome Modification

[0054] In the embodiment, the specific implementation steps of plasmid construction and extraction, cell recovery and culture, tool cell line screening, and cell transfection refer to steps 1-6 of Example 1.

[0055] Verification of 7 small molecule compounds improving the efficiency of genome-directed modification

[0056] At the same time, the optimal concentrations of 4 candidate small molecule compounds Irinotecan, Docetaxel, Miomycin and Nocodazole for HEK293T (human cells), BHK-21 (mouse cells) and PEF cell lines (pig cells) site-directed insertion efficiency were further verified. After the 24-well plate cells in step 6 were cultured for 12 hours, Irinotecan, Docetaxel, Miomycin or Nocodazole with different concentration gradients were added. After continuing to culture for 48 hours, flow cytometry was used to detect the number of green fluorescent cells and th...

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Abstract

The invention relates to the technical field of genetic engineering, in particular to a method for improving the efficiency of genome site-directed modification by using small molecular compounds. A CRISPR / Cas9 system and the small molecular compounds are combined to act on cells to achieve the method. Specifically, the CRISPR / Cas9 system comprises a gRNA fragment of a target gene, the target genecan be identified at a directed site, and the target gene is broken in two chains, and at the break site, the small molecular compounds play a role in homologous recombination repair, the genome site-directed modification is effectively completed, and the efficiency of cell genome site-directed modification treated by the small molecular compounds is significantly improved.

Description

technical field [0001] The disclosure relates to the technical field of genetic engineering, in particular to a method for improving the efficiency of genome-specific modification by using small molecule compounds. Background technique [0002] DNA double-strand breaks (DSBs) that occur naturally in the genome or are artificially introduced are the prerequisites for making site-directed modifications. Traditional gene manipulation techniques utilize donor and naturally occurring DSBs, with an integration efficiency of only 10 5 ~10 7 , which brings great difficulties to the study of genome-directed modification animals. In recent years, with the discovery and application of gene editing tools such as zinc finger endonuclease (ZFN), transcription activator-like effector nuclease (TALEN), clustered regularly interspaced short palindromic repeat (CRISPR), etc. The efficient introduction of DSB into the genome provides an opportunity for the realization of genome-specific mod...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/90
CPCC12N15/907
Inventor 吴珍芳李国玲张献伟王豪强李紫聪蔡更元刘德武杨化强
Owner SOUTH CHINA AGRI UNIV
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