Four sgRNAs designed for human adrb2 gene

A gene and gene knockout technology, applied in DNA/RNA fragments, recombinant DNA technology, genetic engineering, etc., can solve the problems of cytotoxicity, restrict application, increase the difficulty of design and screening, reduce off-target effects and achieve efficient knockout Effect

Active Publication Date: 2021-04-13
江苏集萃崛创生物科技研究所有限公司
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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 for human adrb2 gene
  • Four sgRNAs designed for human adrb2 gene
  • Four sgRNAs designed for human adrb2 gene

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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 sgRNA sequences designed for human ADRB2 gene. Log in to the website to retrieve the CDS sequence of the human ADRB2 gene, and according to the design principles of sgRNA, four sgRNAs were designed in a head-to-head arrangement near the 5' end of the CDS region. On this basis, single sgRNA expression plasmids and multiple sgRNA vectors were constructed respectively. T7EN1 enzyme digestion identification and TA clone sequencing confirmed that the two plasmids can guide the Cas9 protein to cut target genes after transfection into HEK293T cells, especially the gene modification efficiency of multiple sgRNAs can be as high as 100%.

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...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113
CPCC12N15/113C12N2310/10
Inventor 施明郑骏年刘丹孙毓
Owner 江苏集萃崛创生物科技研究所有限公司
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