High-efficiency artificial activating transcription factor dCas9-TV, and coding gene and applications thereof

Abstract

The invention relates to a high-efficiency artificial activating transcription factor dCas9-TV, and a coding gene and applications thereof. According to a construction method, the carboxyl terminal ofnuclease inactivated Cas9 protein (dCas9) is connected with a plurality of copies of VP64 and TAL transcription-activating domains so as to obtain a series of novel artificial activating transcription factors, and obtain dCas9-TV with the best transcriptional activation activity via screening. When only one guide RNA (gRNA) is adopted for targeting a specific gene promoter, dCas9-TV is capable ofrealizing high efficiency activating of transcription of endogenous genes of Arabidopis thaliana and paddy rice; when a plurality of gRNA are adopted for targeting a plurality of target genes, dCas0-TV is capable of realizing transcription activation of a plurality of genes. In addition, it is confirmed that dCas9-TV possesses the same high efficiency targeting transcription activation activity in human cells. An in vitro assembled dCas9-TV / gRNA ribonucleoprotein compound can be adopted for transcription activation of Arabidopis thaliana and paddy rice endogenous genes. The high-efficiency artificial activating transcription factor dCas9-TV can be adopted in the fields such as genome genetic screening, metabolite biosynthesis pathway reconstruction, and crop improvement.

Description

technical field [0001] The invention relates to a high-efficiency artificial transcription activator dCas9-TV and its coding gene and application, belonging to the field of biotechnology, in particular to genetic improvement of crops based on gene overexpression and artificial regulation of eukaryotic cell metabolic pathways. Background technique [0002] Transcription regulation is an important part of eukaryotic gene expression, which can change the level of gene expression by changing the rate of transcription. This process involves the joint participation of many transcriptional regulatory elements. Among them, the transcription activator can promote the interaction between RNA polymerase and the promoter, and increase the transcription level of the target gene. Transcription activator contains DNA-binding domain (DNA-binding domain) and transcription activation domain (transcription activation domain, TAD), the former determines the specificity of transcription activat...

AI Technical Summary

Problems solved by technology

When only a single gRNA is used to bind to the target gene promoter, in most cases the dCas9-VP64 transcriptional activator can only up-regulate the target gene transcription by less than 2 times at most, which cannot meet the needs of target gene transcriptional activation, which greatly limits the application of this technology

But on the one hand, this increases the possibility of off-target, and at the same time increases the complexity of experimental manipulations, and reduces the throughput of the entire transcriptional activation system (i.e., the ability to activate multiple genes simultaneously)

[0006] In summary, there is still a lack of highly efficient artificial transcriptional activators in plants, and it is difficult to achieve effective transcriptional activation of single or multiple endogenous genes.

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