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A method for improving the efficiency of genome-directed modification using small molecule compounds

A small molecular compound and genome fixed-point technology, applied in the field of genetic engineering, can solve the problem of low efficiency of genome fixed-point modification

Active Publication Date: 2021-10-12
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • A method for improving the efficiency of genome-directed modification using small molecule compounds
  • A method for improving the efficiency of genome-directed modification using small molecule compounds
  • A method for improving the efficiency of genome-directed modification using small molecule compounds

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Screening of small molecularcides in which genetic set point modification efficiency

[0032] 1 vector construction

[0033] 1.1 Green Fluorescent Protein (GFP) Report Carrier Gene Synthesis

[0034] The GAPDH-GFP report carrier gene is synthesized by Nanjing Jinsi Biotechnology Co., Ltd., as follows: figure 1 A shows the GAPDH-GFP Report Carrier Mode: CRISPR / CAS9 Cut GAPDH Gene Produces DSB, cells use donor GAPDH-T2A-GFP plasmid or donor GAPDH-T2A-GFPPCR product template HDR repair or NHEJ repair, if Homologous repair occurs, then the green fluorescent protein gene in series can be expressed, and there is no fluorescent protein if homologous repair occurs; figure 1 B. SSODN-GFP Report Carrier Mode: Report Carrier EGFP Sequence Insertion Sequence SEQ ID NO: 1: 5'-gtgag at NO: 1: 5'-gtgag at NO: 1: 5'-gtgag at NO: 1: 5'-gtgag at NO: 1: 5'-gtgagattatctgaccgtaAGG-3 ', CRISPR / CAS9 Cutting DSB, cells As a homologous template with SSODN of 140 nT. Template sequence...

Embodiment 2

[0053] Example 2: Improve the efficiency of genetic set point modification using small molecule compounds

[0054] In the examples, the contents of the sequencing and extraction, the recovery and culture of cells, the tool cell line screening, and the specific embodiment of the cell transfection, and the steps 1-6 of the embodiments will be given.

[0055] 7 Molecular Compound Improve Genome Validation

[0056] At the same time, four candidate small molecule compounds Irinotecan, Docetaxel, Miomycin and Nocodazole were further concentrated on HEK293T (human cells), BHK-21 (mouse cells) and PEF cell lines (pig source cells) fixed-point insertion efficiency. The 24-well plate cells in step 6 continued to cultivate for 12 h, adding different concentrations of Irinotecan, Docetaxel, Miomycin or Nocodazole. After 48 h was continued for 48 h, the amount of cellular fixed-point modification efficiencies in the amount of green fluorescent cells were detected using a flow cytometry. Such a...

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Abstract

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. The method is realized by co-acting the CRISPR / Cas9 system and small molecule compounds on cells. Specifically, the CRISPR / Cas9 system contains a gRNA fragment of the target gene, which can identify the target gene at a fixed point and cause a double-strand break in the target gene. At the break, small molecular compounds play a role, perform homologous recombination repair, and efficiently complete the genome. Site-directed modification, the efficiency of site-directed modification of cell genomes treated with small molecule compounds is significantly improved.

Description

Technical field [0001] The present disclosure relates to the field of genetic engineering techniques, and in particular, to a method of improving the genetic component modification efficiency using a small molecule compound. Background technique [0002] The genome natural production or human introduction DNA double strand break (DSB) is the premise of preparing fixed-point modification. Traditional genetic operation technology utilizes donor and natural DSB, integrated efficiency is only 10 5 ~ 10 7 It has brought great difficulties to the study of genotype point modified animals. In recent years, with the discovery and application of gene editing tools such as gene editing tools such as nucleic acid endonuclease (ZFN), transcriptional activation factor effectaccoase (TALEN), regular clusters, short-term dispersion (CRISPR), etc. Efficiently introduce DSB in the genome, providing an opportunity for genetic settles. The CRISPR / CAS9 technique gets rid of synthesis and assembly o...

Claims

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Application Information

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Patent Type & Authority Patents(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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