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A sgRNA combination targeting ahrr gene and its application

A gene-targeted technology, applied in the field of gene editing, can solve the problems of difficulty in meeting the needs of obesity research, poor obesity phenotype stability, lack of unified modeling standards, etc., to achieve stability and heritability, and high gene editing efficiency. , the effect of wide application value

Active Publication Date: 2022-07-08
新开源晶锐广州生物医药科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, increasing the weight of rats by eating is greatly affected by seasonal, environmental and individual differences, and the repeatability is poor. The obesity phenotype is not stable and cannot be passed on to offspring, so it has great limitations.
[0004] At present, the construction methods of obesity animal models generally have cumbersome modeling conditions, long cycle, poor stability of obesity phenotype, lack of unified modeling standards, and it is difficult to meet the research needs of different pathogenesis of obesity. How to provide a method with simple construction conditions and cycle time The construction method of short, obese phenotype stable and reproducible obesity model has become an urgent problem to be solved

Method used

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  • A sgRNA combination targeting ahrr gene and its application
  • A sgRNA combination targeting ahrr gene and its application
  • A sgRNA combination targeting ahrr gene and its application

Examples

Experimental program
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Embodiment 1

[0073] The present embodiment provides an sgRNA combination targeting the AHRR gene, the sgRNA combination targeting the AHRR gene includes sgRNA1 and sgRNA2, the sgRNA1 includes the nucleic acid sequence shown in SEQ ID No. 1, and the sgRNA2 includes SEQ ID No. 1. 2 shows the nucleic acid sequence.

[0074] SEQ ID No. 1: GGAGATNTCGCCAAGTNCATGGG;

[0075] SEQ ID No. 2: GGCACATCTANCGTNATTATTGG;

[0076] Wherein, N represents any one of A, T, C or G.

[0077] The sgRNA1 specifically targets intron 5 of the AHRR gene, and the sgRNA2 specifically targets intron 8 of the AHRR gene, with good specificity, low off-target rate, and wide application value.

Embodiment 2

[0079] This embodiment provides an AHRR gene editing system, the AHRR gene editing system includes sgRNA targeting AHRR gene in combination of sgRNA1 and sgRNA2 and mRNA of Cas9 nuclease.

[0080] The AHRR gene editing system has the ability to knock out the AHRR gene in the genome. Through the cooperation of the sgRNA combination, it can reduce the off-target rate, reduce the probability of non-specific editing, and has the ability to knock out large fragments, and the editing efficiency is higher; Cas9 is selected. The mRNA of nuclease is less toxic to cells, and the edited individuals are more likely to survive, reducing the difficulty of screening.

Embodiment 3

[0082] This example provides a recombinant cell, which is a fertilized egg cell of a C57BL / 6 mouse whose genome has been mutated in the AHRR gene after being edited by the AHRR gene editing system in Example 2.

[0083] The recombinant cells are constructed by the following methods:

[0084] (1) In vitro transcribe the mRNA of the Cas9 nuclease gene, and mix it with the sgRNA targeting the AHRR gene to obtain the AHRR gene editing system;

[0085] Ovulation induction was performed on C57BL / 6 mice, and fertilized eggs were cultured after in vitro fertilization;

[0086] (2) Microinjecting the AHRR gene editing system into the nucleus of a C57BL / 6 mouse fertilized egg cell to obtain a recombinant cell.

[0087] By directly injecting the mRNAs of sgRNA1, sgRNA2 and Cas9 nucleases into the nucleus of fertilized egg cells, the efficiency of gene editing is improved; fertilized eggs are selected to construct recombinant cells, and sense mutations can be inherited through cell divis...

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Abstract

The present invention provides a combination of sgRNA targeting AHRR gene and application thereof, the combination of sgRNA targeting AHRR gene includes sgRNA1 and / or sgRNA2, and the sgRNA1 includes the nucleic acid sequence shown in SEQ ID No. 1, and the sgRNA2 includes the nucleic acid sequence shown in SEQ ID No.2. The present invention also provides an AHRR gene editing system, a recombinant cell and a method for constructing the same, and a method for constructing an obese animal model. Obesity signs, larger body size, heavier body weight, and higher body fat content can be used in the screening of hypoglycemic and lipid-lowering related drugs, and have extremely wide application value.

Description

technical field [0001] The invention belongs to the technical field of gene editing, and in particular relates to a sgRNA combination targeting AHRR gene and its application. Background technique [0002] With the changes in people's dietary structure and lifestyle, obesity has become an important health problem facing the world today. Obesity is a complex metabolic system disease, which can cause related metabolic disorders and diseases, such as cardiovascular and cerebrovascular diseases, lipid metabolism disorders, insulin resistance, type II diabetes and metabolic syndrome. In clinical practice, simple obesity is the main disease, which is characterized by no obvious changes in the morphology and function of the nervous and endocrine systems, but accompanied by disorders of lipid and glucose metabolism. [0003] To study the development mechanism of obesity and explore its treatment methods is the premise to guide the formulation of obesity prevention and treatment stra...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/89C12N15/873C12N5/10C12N15/55C12N15/12A61K49/00
CPCC12N15/113C12N15/89C12N15/873C12N9/22C07K14/4703C12N5/0604A61K49/0008C12N2800/107C12N2510/00C12N2310/20A01K67/0276A01K2227/105A01K2267/0362Y02P60/87
Inventor 钟诗龙汪静朱茜陈爽吴圆圆汤雅男
Owner 新开源晶锐广州生物医药科技有限公司
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