Method for specifically knocking out fumarylacetoacetate hydrolase (FAH) gene by using CRISPR-Cas9 and specific sgRNA

A specific and genetic technology, applied in the field of gene knockout and genetic engineering, to achieve the effect of improving efficiency

Pending Publication Date: 2020-05-05
立沃生物科技(深圳)有限公司
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0005] At present, there have been reports on the knockout of FAH gene in small model animals such as rats and mouse models, and the existing methods have not yet been used to knock out the FAH gene by CRISPR / Cas9 in rabbit and pig models.

Method used

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  • Method for specifically knocking out fumarylacetoacetate hydrolase (FAH) gene by using CRISPR-Cas9 and specific sgRNA
  • Method for specifically knocking out fumarylacetoacetate hydrolase (FAH) gene by using CRISPR-Cas9 and specific sgRNA
  • Method for specifically knocking out fumarylacetoacetate hydrolase (FAH) gene by using CRISPR-Cas9 and specific sgRNA

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Embodiment Construction

[0043] The realization of the purpose of the present invention, functional characteristics and advantages will be further described in conjunction with the embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

[0044] The present invention provides a specific sgRNA for specifically targeting pig and rabbit FAH genes,

[0045] (1) The target sequence of the sgRNA on the pig and rabbit FAH genes conforms to the sequence arrangement of 5'-N(20)NGG-3',

[0046] Wherein N(20) represents 20 consecutive bases, wherein each N represents A or T or C or G, and the target sequence conforming to the law can be located in the sense strand or the antisense strand of the DNA sequence;

[0047] (2) The target sequences of the sgRNA on the pig and rabbit FAH genes are respectively located in the 5 exons coding regions of the N-terminal of the pig and rabbit FAH genes, or the main p...

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Abstract

The invention relates to a method for specifically knocking out a fumarylacetoacetate hydrolase (FAH) gene by using CRISPR-Cas9 and specific sgRNA. The method is applied to specifically knock out theFAH gene of pigs and rabbits and comprises the following specific steps: S1: selection and design of a sgRNA target sequence; S2: construction of a CRISPR-Cas9 vector of the FAH gene; S3: obtaining ofa pseudotype lentivirus expressing FAH sgRNA and Cas9 protein; and S4: infection of a target cell and detection of an FAH gene knockout effect. The target sequence of the specific sgRNA on the FAH genes of the pigs and rabbits conforms to a sequence arrangement law of 5'-N(20)NGG-3'. The specific sgRNA is applied to the method for specifically knocking out the FAH gene of the pigs and rabbits byusing the CRISPR-Cas9, can rapidly, accurately, efficiently and specifically knock out the FAH gene of the pigs and rabbits respectively, and effectively solves the technical problems of long period and high cost in construction of pigs and rabbits with the FAH gene knocked out.

Description

【Technical field】 [0001] The present invention relates to the technical field of genetic engineering, in particular to the technical field of gene knockout, in particular to a method for specifically knocking out pig and rabbit FAH genes by CRISPR-Cas9 and a specific sgRNA for specifically targeting pig and rabbit FAH genes. 【Background technique】 [0002] Human primary hepatocytes refer to hepatocytes cultured immediately after removal from human liver tissue, which can be used for basic life science research, drug evaluation, cell transplantation and liver 3D printing. However, liver cells mainly come from surgical resection of liver tissue, voluntary donation of remains, etc. Due to the ethical problems of these materials, and the traditional Chinese culture, the rate of liver or liver tissue donation is relatively low in the world. In vivo expansion of hepatocytes is a feasible solution that maintains the differentiated state of hepatocytes while obtaining a substantial ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/90C12N15/867C12N15/113
CPCC12N15/907C12N15/86C12N15/1137C12N9/14C12Y307/01002C12N2800/107C12N2810/10C12N2740/15043C12N2310/10C12N2310/20
Inventor 周明
Owner 立沃生物科技(深圳)有限公司
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