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Construction method of recombinant sgRNA backbone vector in CRIPSR/Cas9 system

A technology of a skeleton carrier and a construction method, which is applied in the fields of molecular biology and biology, and can solve the problems of low positive rate of bacterial strains, high requirements for operation and reagent preparation, time-consuming and laborious selection of positive bacterial strains, etc.

Active Publication Date: 2021-05-14
JIANGNAN UNIV
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Problems solved by technology

[0004] In view of the above problems, the present invention provides a method for constructing a recombinant sgRNA backbone carrier in the CRIPSR / Cas9 system, which can solve the operational requirements of the existing construction method and the relatively high requirements for reagent preparation, and the positive rate of bacterial strains obtained by ordinary technicians is low. Picking out time-consuming technical issues

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  • Construction method of recombinant sgRNA backbone vector in CRIPSR/Cas9 system
  • Construction method of recombinant sgRNA backbone vector in CRIPSR/Cas9 system
  • Construction method of recombinant sgRNA backbone vector in CRIPSR/Cas9 system

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Embodiment 1

[0024] The construction method of recombinant sgRNA backbone carrier in CRIPSR / Cas9 system, it comprises the following steps:

[0025] (1) Preparation of linearized sgRNA empty plasmid

[0026] The sgRNA empty plasmid used has a pair of symmetrical IIS-type endonuclease cutting sites, the purpose is to allow the short sgRNA fragment to be seamlessly inserted between the promoter and the scaffold; if there is an sgRNA empty plasmid, directly use the corresponding IIS Type endonuclease digestion treatment is sufficient; after the digestion is completed, the gel is recovered to obtain a linearized sgRNA empty plasmid.

[0027] If there is no sgRNA empty plasmid, but only a plasmid that has been constructed into a certain sgRNA, then design primers to perform full-plasmid PCR on this plasmid to eliminate the sgRNA on the plasmid, and obtain an sgRNA empty plasmid containing an IIS-type endonuclease enzyme-cut linker; The initial sgRNA plasmid cannot have IIS-type endonuclease rec...

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Abstract

The present invention provides a method for constructing a recombinant sgRNA backbone carrier in the CRIPSR / Cas9 system, which can solve the operational requirements of the existing construction method and the relatively high requirements for reagent preparation. The positive rate of strains obtained by ordinary technicians is low, and the selection of positive strains is time-consuming and laborious. technical problem. A method for constructing a recombinant sgRNA backbone vector in a CRIPSR / Cas9 system, comprising the following steps: (1) preparing a linearized sgRNA empty plasmid; (2) preparing a distinguishing fragment; (3) preparing a recombinant sgRNA backbone vector; (4) selecting a recombinant sgRNA backbone carrier; characterized in that: the distinguishing fragment of the step (2) includes sacB Genes and their open reading frames (ORFs), promoters and terminators.

Description

technical field [0001] The invention relates to the fields of molecular biology and biotechnology, in particular to a method for constructing a recombinant sgRNA backbone vector in a CRIPSR / Cas9 system. Background technique [0002] The CRIPSR / Cas9 system, known as the third-generation gene editing technology, is widely used in the genetic modification of various in vivo and in vitro systems, the construction of transgenic model animals, and even the field of gene therapy. When using the CRIPSR / Cas9 system for gene editing operations, it is necessary to select a supply form of sgRNA. Taking the gene editing of mammalian cells as an example, plasmids or viruses can be used as vectors to express sgRNA, or sgRNA can be produced and purified in vitro, and then directly transfected into cells to perform functions. Of course, the form of introducing sgRNA depends on the needs of the experiment and the characteristics of the cell itself, but in general, it is more economical and c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N15/66
CPCC12N15/66C12N15/70C12N2800/80C12N2810/10
Inventor 戴晓峰高雄孙曼曼白仲虎
Owner JIANGNAN UNIV