Establishing method of sgRNA expression component driven by gene editing U6 promoter

Abstract

The invention discloses an establishing method of a sgRNA expression component driven by a gene editing U6 promoter, and belongs to biogenetic engineering. By using an IDH1 gene of a human genome as atarget gene for CRISPR / Cas9 gene editing and designing the specific sequence of the gene in sgRNA, the complete sequence of the sgRNA expression component with the U6 promoter is obtained, the U6-sgRNA expression component is amplified, a PCR primer of the U6-sgRNA expression component is synthesized, PAGE purification is conducted, and finally the step of overlapping PCR to obtain a complete double-chain DNA fragment of the U6-sgRNA expression component is conducted for completing establishment; the whole establishing method is high in gene editing success rate, convenient to use and low incost.

Description

technical field [0001] The invention relates to the technical field of biogenetic engineering, in particular to a method for constructing a gene editing U6 promoter-driven sgRNA expression module. Background technique [0002] Gene editing is one of the most popular scientific research and treatment methods today. It can modify and edit specific gene sequences. It has been widely used in scientific research for gene function research, model animal construction, and clinical treatment of diseases. The current main systems for gene editing include ZFN, TALEN and CRISPR / Cas9 systems. Since the ZFN and TALEN systems need to reconstruct the protein according to the target sequence of gene editing, it takes a lot of time and cost, while the CRISPR / Cas9 system only needs to reconstruct the short-chain RNA sequence according to the sequence of the target gene. It has the advantages of timeliness and cost. In the CRISPR / Cas9 system, the Cas9 nuclease needs the assistance of crRNA a...

AI Technical Summary

Problems solved by technology

Although this method is relatively fast, since the synthesized fragment does not contain a type III RNA polymerase promoter and relies on the type III RNA polymerase promoter on the vector, it is necessary to use a vector containing a type III RNA polymerase promoter when selecting a vector , the lack of flexibility in the selection of the vector, the replacement of the vector and the system is more cumbersome, and the possibility of reverse connection may occur when connecting into the vector plasmid, which requires further screening and inspection, and the efficiency is low

The second technical solution, that is, the whole gene synthesis of sgRNA expression components containing type III RNA polymerase promoter, this method is relatively straightforward, but very expensive, if you need to construct sgRNA expression components for a large number of different sites, it will cost a lot financial resources

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