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Specie limitation-free eucaryote gene targeting method having no bio-safety influence and helical-structure DNA sequence

A biosafety and gene targeting technology, applied in the field of genetic engineering, can solve problems such as large limitations, time-consuming, labor-intensive and cost-intensive problems, and achieve the effects of high accuracy, cost reduction, and low cost

Active Publication Date: 2013-08-07
NANJING SYNC BIOTECH
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Problems solved by technology

[0007] Aiming at the problems of large limitations, time-consuming, labor-intensive and high cost in realizing specific genome modification through ZFNs or TALENs in the prior art, the present invention provides a kind of eukaryotic gene targeting without species limitation and without biological safety problems Method and helical DNA sequence, the method has simple steps, flexible recognition sites and low consumption

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  • Specie limitation-free eucaryote gene targeting method having no bio-safety influence and helical-structure DNA sequence
  • Specie limitation-free eucaryote gene targeting method having no bio-safety influence and helical-structure DNA sequence
  • Specie limitation-free eucaryote gene targeting method having no bio-safety influence and helical-structure DNA sequence

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Embodiment 1

[0037] A eukaryotic gene targeting method without species limitation and without biological safety problems in this embodiment, the steps are:

[0038] (1) Design and construction of CRISPR / Cas9 and chimeric RNA

[0039] 1) Optimize the codon encoding Cas9

[0040]Cas9 belongs to the second type of CRISPR / Cas system, which is derived from prokaryotic cells. In order to make it better expressed in eukaryotic cells, the Cas9 coding sequence was first optimized and used in eukaryotic cells without changing amino acids. Codons encoding amino acids;

[0041] 2) Cas9 nuclease vector construction and Cas9 nuclease mRNA synthesis

[0042] According to the optimized sequence in step 1), custom-synthesize the DNA sequence encoding Cas9 nuclease, and insert it into the following expression vectors: pST1374-Cas9, pST1374-NLS-Flag-Cas9 (this vector contains the vector pST1374 -In addition to all the sequences of Cas9, there is a sequence NLS encoding the nucleus localization signal and ...

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Abstract

The invention discloses a specie limitation-free eucaryote gene targeting method having no bio-safety influence and a helical-structure DNA sequence, and belongs to the field of gene engineering. The specie limitation-free eucaryote gene targeting method comprises the following steps of 1, designing and constructing CRISPR / Cas9 and chimeric RNA, and 2, carrying out Cas9mRNA internal translation so that Cas9 nuclease and the chimeric RNA are bonded, carrying out fixed point clipping so that DNA double-chain cleavage is realized after the clipping, and introducing an exogenous DNA by induction of a natural DNA restoration process which is a non-homologous end bonding process of cells so that cell endogenous gene modification is realized. The specie limitation-free eucaryote gene targeting method has simple processes, realizes flexible site recognition and has low energy consumption.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and more specifically relates to a eukaryotic gene targeting method and a helical structure DNA sequence without species limitation and without biological safety problems. Background technique [0002] Gene targeting technology is an experimental method for directional alteration of the genetic information of an organism. In recent years, with the development of biotechnology, zinc finger nuclease (ZFN) technology and transcription activator-like effector nuclease (TALEN) technology have been applied in gene targeting, and these two technologies have greatly improved the specificity of targeted modification. ZFN is composed of a DNA recognition domain and a non-specific endonuclease. The DNA recognition domain is composed of a series of Cys2-His2 zinc-finger proteins (zinc-fingers) in series (generally 3-4), and each zinc-finger protein recognizes and binds a specific triplet base. Multiple z...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/55C12N15/11C12P19/34
Inventor 沈彬黄行许
Owner NANJING SYNC BIOTECH
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