Dual gRNA (guide Ribonucleic Acid) gene expression element and construction method and application thereof

Abstract

The invention discloses a dual gRNA (guide Ribonucleic Acid) gene expression element and a construction method and an application thereof, and relates to the technical field of gRNA gene engineering.The gRNA gene expression element comprises the following structure: polyclonal restriction enzyme cutting site I-U6 promoter-gRNA specific sequence 1-structure sequence-intermediate connection sequence-U6 promoter-gRNA specific sequence 2-structure sequence-polyclonal restriction enzyme cutting site III. Two ends of the designed dual gRNA whole-set gene expression element are independently provided with a polyclonal restriction enzyme cutting site, the polyclonal restriction enzyme cutting sites can be convenient to be connected with subsequent expression vectors, and the expression vectors are not restricted. Therefore, the dual gRNA whole-set gene expression element is directly connected to a Cas9 nuclease expression vector to further improve transfection efficiency and a gene editing success rate, the polyclonal restriction enzyme cutting sites are in the presence in the middle and can be used for splitting a dual gRNA system into two single gRNA systems, and therefore, various animal models are constructed.

Description

Technical field [0001] The present invention relates to the technical field of gRNA genetic engineering, in particular to a double gRNA gene expression element and its construction method and application. Background technique [0002] Gene editing is one of the hotspots of current research. Among them, CRISPR / Cas9 has quickly become popular around the world because of its simple and convenient operation, low time and cost advantages. In the CRISPR / Cas9 system, Cas9 nuclease needs the assistance of crRNA and tracrRNA to recognize the target sequence, and then cut the target sequence to form a broken double strand for gene editing. After modification, crRNA and tracrRNA are fused into one RNA molecule, namely gRNA. [0003] In the CRISPR / Cas9 system, Cas9 nuclease can perform single-site gene editing with the assistance of one gRNA, or multi-site gene editing with the assistance of multiple gRNAs. Single-point editing is the most common at present, but single-point gene editing ca...

AI Technical Summary

Problems solved by technology

[0004] The applicant's research on the existing two-site gene editing method found that due to the limitation of the carrier, the commonly used two-site gene editing has the problems of low transfection success rate and inconsistent expression levels, which will bring time and effort for screening. Increase

At the same time, due to the need to synthesize multiple longer fragments, the cost is greatly increased

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