An artificial transcription activator dcas9-tv and its encoding gene and application

Abstract

The invention relates to a high-efficiency artificial transcription activator dCas9‑TV, its coding gene and its application. In the present invention, multiple copies of VP64 and TAL transcriptional activation domains are connected to the carboxy-terminal of the nuclease-inactivated Cas9 protein (dCas9) to generate a series of novel artificial transcriptional activators, and dCas9 with the best transcriptional activation activity is screened out -TV. When only one guide RNA (gRNA) was used to target specific gene promoters, dCas9‑TV efficiently activated transcription of endogenous genes in Arabidopsis and rice. When multiple gRNAs are used to target multiple target genes, dCas9‑TV can simultaneously achieve transcriptional activation of multiple genes. In addition, dCas9‑TV was also confirmed to have highly efficient targeted transcriptional activation activity in human cells. The dCas9‑TV / gRNA ribonucleoprotein complex assembled in vitro can also activate the transcriptional activation of Arabidopsis and rice endogenous genes. Based on the above excellent activities, it can be applied to genome genetic screening, biosynthesis pathway modification of metabolites, 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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