SgRNA combination of targeted AHRR gene and application of sgRNA combination

A gene and targeting technology, applied in the field of gene editing, can solve problems such as difficulty in meeting the needs of obesity research, lack of unified modeling standards, and poor stability of obesity phenotypes, achieving stability and heritability, and high gene editing efficiency , the effect of extensive application value

Active Publication Date: 2022-01-11
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

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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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  • SgRNA combination of targeted AHRR gene and application of sgRNA combination
  • SgRNA combination of targeted AHRR gene and application of sgRNA combination
  • SgRNA combination of targeted AHRR gene and application of sgRNA combination

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Embodiment 1

[0073] This embodiment provides a combination of sgRNAs targeting AHRR genes, the combination of sgRNAs targeting AHRR genes includes sgRNA1 and sgRNA2, the sgRNA1 includes the nucleic acid sequence shown in SEQ ID No.1, and the sgRNA2 includes SEQ ID No. The nucleic acid sequence shown in 2.

[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 the sgRNA combination sgRNA1 and sgRNA2 targeting the AHRR gene and the mRNA of the Cas9 nuclease.

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

Embodiment 3

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

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

[0084] (1) Transcribe the mRNA of the Cas9 nuclease gene in vitro, mix it with the sgRNA targeting the AHRR gene, and 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 mRNA of sgRNA1, sgRNA2 and Cas9 nuclease into the nucleus of the fertilized egg cell, the efficiency of gene editing is improved; the fertilized egg is selected to construct recombinant cells, and the sense mutation can be inherited through cel...

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Abstract

The invention provides a sgRNA combination of a targeted AHRR gene and application of the sgRNA combination, the sgRNA combination of the targeted AHRR gene comprises sgRNA1 and / or sgRNA2, the sgRNA1 comprises a nucleic acid sequence as shown in SEQ ID No.1, and the sgRNA2 comprises a nucleic acid sequence as shown in SEQ ID No.2. The invention also provides an AHRR gene editing system, a recombinant cell and a construction method thereof, and a construction method of an obese animal model, through gene editing and screening, the obtained homozygous mouse without the AHRR gene has typical obesity signs, larger body type, heavier body weight, higher fat content in vivo, and can be used for screening blood sugar and lipid reducing related drugs, and has extremely wide application value.

Description

technical field [0001] The invention belongs to the technical field of gene editing, and in particular relates to an sgRNA combination targeting AHRR gene and its application. Background technique [0002] With the changes in people's diet structure and lifestyle, obesity has become an important health problem facing the world today. Obesity is a complex metabolic system disease that can cause related metabolic disorders and diseases, such as cardiovascular and cerebrovascular diseases, lipid metabolism disorders, insulin resistance, type II diabetes and metabolic syndrome. Clinically, it is mainly simple obesity, which is characterized by no obvious changes in the morphology and function of the nervous and endocrine systems, but accompanied by lipid and glucose metabolic process disorders. [0003] Studying the development mechanism of obesity and exploring its treatment methods are the prerequisites for guiding the development of obesity prevention and treatment strategie...

Claims

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Application Information

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Patent Type & Authority Applications(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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