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Four sgRNAs designed by aiming at human source ADRB2 genes

A gene, human-derived technology, used in DNA/RNA fragments, recombinant DNA technology, genetic engineering, etc., can solve problems such as restricting application, cytotoxicity, increasing design, and screening difficulty, achieving efficient knockout and reducing off-target effects. Effect

Active Publication Date: 2018-09-18
XUZHOU MEDICAL UNIV
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Problems solved by technology

On the one hand, there is context sequence dependence in the recognition domain of zinc finger nucleases, which increases the difficulty of design and screening; on the other hand, the off-target cleavage of zinc finger nucleases will lead to cytotoxicity, which restricts its application in the field of gene therapy

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  • Four sgRNAs designed by aiming at human source ADRB2 genes
  • Four sgRNAs designed by aiming at human source ADRB2 genes
  • Four sgRNAs designed by aiming at human source ADRB2 genes

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Embodiment Construction

[0031] The technical scheme of this patent will be further described in detail below, and the parts of the technical features described in the present invention that are not described in detail all adopt the existing mature technology.

[0032] The present invention will be described in further detail below in conjunction with the accompanying drawings.

[0033] The present invention aims at four sgRNAs designed for the human ADRB2 gene. First, you need to log in to the UCSC GenomeBrowser Home website to retrieve the CDS sequence of the human ADRB2 gene. Then, according to the design principles of sgRNA, a head-to-head design was adopted at the 5' end of its CDS region, four target sites were selected, and four sgRNAs were designed (such as figure 1 ). By connecting with the pGL3-U6-sgRNA-PGK-puromycin vector, a single sgRNA expression vector targeting the ADRB2 gene was constructed, and it was confirmed by T7EN1 enzyme digestion that the four sgRNAs could effectively guide ...

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Abstract

The invention belongs to the technical field of gene modification, and discloses four sgRNAs designed by aiming at human source ADRB2 genes. The CDS sequence of the human source ADRB2 genes is retrieved by website login; according to the sgRNA design principle, the end-to-end arrangement mode is used as a near 5' end of a CDS region; the four sgRNAs are designed. On the basis, single sgRNA expression plasmids and multiplex sgRNA carriers are respectively built. Through T7EN1 digestion identification and TA clone sequencing, the effect that the two kinds of plasmids can guide Cas9 protein to cut target genes after the HEK293T cell transfection; particularly, the multiplex sgRNA gene modification efficiency is as high as 100 percent is confirmed.

Description

technical field [0001] The invention belongs to the technical field of CRISPR / Cas9 gene editing, and specifically refers to four sgRNAs designed for human ADRB2 gene. Background technique [0002] Gene editing technology is a technology developed in recent years that can precisely modify genes. In the field of scientific research, gene editing technology can be used for the rapid construction of model organisms; in the field of agriculture, this technology can be used to transform plant varieties; and in the field of health care, this technology may also achieve the purpose of treating diseases by modifying human genes . Therefore, gene editing technology has extremely broad development prospects and application value. [0003] At present, gene editing technologies mainly include zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeat technology (CRISPR / Cas9). In terms of technical pri...

Claims

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Application Information

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IPC IPC(8): C12N15/113
CPCC12N15/113C12N2310/10
Inventor 施明郑骏年刘丹孙毓
Owner XUZHOU MEDICAL UNIV
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