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Establishment method of T7-RNA-polymerase-mediated CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing system

A gene editing and polymerase technology, applied in the direction of recombinant DNA technology, introduction of foreign genetic material using vectors, etc., can solve the problems of high difficulty, not widely popularized and used, and high cost, and achieves quick and easy use, convenient construction, and simplified design steps. Effect

Inactive Publication Date: 2016-08-17
NORTHWEST A & F UNIV
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Problems solved by technology

However, chemical synthesis of full-length crRNA: tracrRNA is difficult and expensive, and is not suitable for widespread use

Method used

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  • Establishment method of T7-RNA-polymerase-mediated CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing system
  • Establishment method of T7-RNA-polymerase-mediated CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing system
  • Establishment method of T7-RNA-polymerase-mediated CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing system

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Embodiment 1

[0029] A method for gene editing using the CRISPR / Cas9 gene editing system, the method is based on the highly specific recognition principle of T7 RNA polymerase and T7 promoter, first constructing T7 promoter-sgRNA, expressing T7 promoter-sgRNA and Cas9 The plasmid and the T7 RNA polymerase expression plasmid are jointly transferred into the cell, and the T7 RNA polymerase catalyzes the T7 promoter to initiate the transcription of sgRNA, thereby guiding Cas9 to cut at the target site and complete gene editing.

[0030] T7 RNA polymerase is an RNA polymerase that specifically catalyzes the formation of RNA in the 5'-3' direction; T7 RNA polymerase has a high degree of promoter specificity, and only transcribes the DNA fragment or DNA located downstream of the T7 promoter in T7 phage replica.

[0031] The T7 promoter is the mainstream promoter of the E. coli expression system today. The promoter is powerful and specific, and it is the preferred promoter for prokaryotic expressi...

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Abstract

The invention mainly belongs to the technical field of higher organism genome editing, and particularly relates to an establishment method of a T7-RNA-polymerase-mediated CRISPR (clustered regularly interspaced short palindromic repeats) / Cas9 gene editing system. The method is based on the highly specific recognition principle for the T7 RNA polymerase and T7 promoter, and comprises the following steps: establishing T7 promoter-sgRNA, transforming the T7 promoter-sgRNA, a Cas9 expression plasmid and a T7 RNA polymerase expression plasmid into cells, and catalyzing the T7 promoter to promote the transcription of sgRNA by using the T7 RNA polymerase so as to guide the Cas9 to perform cutting at the target, thereby completing the gene editing process. By using the T7 promoter to express the sgRNA, the method simplifies the design steps of the CRISPR / Cas9 system, so that more people can quickly and easily research the interested gene locus by using the CRISPR / Cas9 gene editing tool.

Description

technical field [0001] The invention mainly belongs to the technical field of genome editing of higher organisms, and in particular relates to a method for constructing a T7 RNA polymerase-mediated CRISPR / Cas9 gene editing system. Background technique [0002] The discovery and modification of gene editing tools targeting higher animal genome loci are of great significance to the research and application of genetic engineering and genetic engineering. The genetic network of higher organisms is intricate, and a simple and efficient tool for genome editing in higher organisms can be modified to provide great help for genetic research in higher organisms. In recent years, the type II CRISPR / Cas9 system found in bacteria is being widely studied and applied. [0003] The CRISPR / Cas9 system is composed of Cas9 endonuclease and single guide RNA (sgRNA). The sgRNA has a 20bp guide sequence that matches the genomic target sequence. [0004] The bacterial type II CRISPR / Cas system i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/65
Inventor 张智英邢佳妮闫强徐坤
Owner NORTHWEST A & F UNIV
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