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Inhibitors for improving the efficiency of genome-directed integration and their applications

A genome-targeted and inhibitor technology, applied in the field of genetic engineering, can solve the problem of low genome-targeted integration efficiency, and achieve the effects of improving the site-targeted integration efficiency, efficiency, and probability.

Active Publication Date: 2021-10-15
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide a kind of inhibitor and application thereof that improves the efficiency of genome-specific integration, to solve the problem of low efficiency of genome-specific integration and improve the efficiency of genome-specific integration

Method used

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  • Inhibitors for improving the efficiency of genome-directed integration and their applications
  • Inhibitors for improving the efficiency of genome-directed integration and their applications
  • Inhibitors for improving the efficiency of genome-directed integration and their applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Construction of CRISPR / Cas9 system and EGFP reporter vector

[0034] EGFP reporter carrier: Two kinds of ACTB-T were constructed separately 2 A-GFP (nucleotide sequence shown in SEQ ID No: 3) and GAPDH-T 2 A-GFP (nucleotide sequence shown in SEQ ID No: 4) reporter carrier, these two kinds of reporter carriers are all synthesized by Nanjing GenScript Biotechnology Co., Ltd., and its working principle is as follows figure 1 Shown: CRISPR / Cas9 system cuts ACTB gene (NC_000007.14) or GAPDH gene (NC_000012.12). After DNA double-strand breaks, cells undergo homologous directional integration, EGFP is integrated into ACTB or GAPDH genes, and cells can produce green fluorescence. The green fluorescent signal can be detected by flow cytometry. Thus, the efficiency of genome-specific integration can be assessed by detecting the number of cells expressing green fluorescence.

[0035]CRISPR / Cas9 system construction: Design the CRISPR / Cas9 guide sequence sgRNA of ACTB...

Embodiment 2

[0040] Embodiment two: the culture of human kidney epithelial cell (293T)

[0041] Adjust the temperature of the water bath to 37°C in advance, take out the frozen cells from the liquid nitrogen, immediately put them into the water bath and shake them quickly to completely dissolve the cell solution within 1-2 minutes. Then add 1ml of cell solution into 8ml of complete medium and mix well, then transfer to 10cm culture dish, and return to 37°C, 5% CO 2 and cultured in an incubator with 95% relative humidity. Subsequent experiments can be carried out when the cells to be cultured grow to 70%-80% confluence.

Embodiment 3

[0042] Example 3: Screening of Small Molecule Compound Library for Appearance Modification to Improve Site-Directed Integration Efficiency

[0043] Small molecule compound library Epigenetics Compound Library was purchased from MCE Company. Use ACTB-T2A-GFP and GAPDH-T2A-GFP reporter vectors to screen whether the 182 compounds in the library can significantly improve the efficiency of genome-specific integration. The specific screening method is as follows: 293T cells are grown to confluence in a 10cm cell culture dish At 70%-80%, transfect the CRISPR / Cas9 vector and EGFP reporter vector according to the instructions of Lipofectamine3000 transfection reagent, and then spread the cells evenly in a 24-well plate. After 12 hours, the old medium was sucked off, and each well was added with 1 μl of small molecule compound (one of 182 compounds in the library) in 1 ml of cell culture medium; control group used 1 μl of DMSO. The cells were then incubated in a 37°C incubator for 48 h...

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Abstract

The invention discloses an inhibitor capable of improving genome site-specific integration efficiency through screening, including at least one of histone deacetylase inhibitors or JAK inhibitors. Histone deacetylase inhibitors can inhibit the deacetylation of histones and promote the acetylation of histones during genome-specific integration, which in turn facilitates the dissociation of DNA from histone octamers and the relaxation of nucleosome structure , so that various transcription factors and cooperative transcription factors can specifically bind to DNA binding sites, activate gene transcription and site-specific integration efficiency. JAK inhibitors can inhibit the spontaneous phosphorylation of cellular JAK, affect its binding to STAT protein, prevent the phosphorylation of STAT transcription factors from being effectively transferred to the nucleus, and affect the binding of non-DNA junction factors, which is beneficial to the binding of NHEJ / HDR factors. Effectively increase the probability of gene integration into the genome and improve the efficiency of genome-specific integration.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a reagent for improving genome-specific integration efficiency and its application. Background technique [0002] Gene editing technology is a technology for precise point-to-point modification of the genome. It can knock out, add, and replace specific DNA fragments, thereby performing precise gene editing at the genome level. The essence of the technology is to use non-homologous end-joining repair and homologous recombination repair, combined with specific DNA target recognition and DNA sequence changes completed by endonucleases. In the field of medicine, researchers build model animals to gain an in-depth understanding of the pathogenesis of diseases, explore gene functions, transplant xenogeneic organs, and transform related disease genes to achieve the purpose of gene therapy, etc.; in the field of animal breeding, researchers use gene editing technology Knock ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/90C12N15/85C12N15/65C12N5/10C12Q1/02C12Q1/6897
CPCC12N5/0686C12N15/65C12N15/85C12N15/907C12N2501/065C12N2501/727C12N2503/00C12N2510/00C12N2800/107C12Q1/6897G01N33/5008
Inventor 吴珍芳许春辉李国玲张献伟李紫聪蔡更元郑恩琴杨化强
Owner SOUTH CHINA AGRI UNIV
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