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

A technology of transcription activator and transcription activation, applied in the direction of genetic engineering, using vectors to introduce foreign genetic material, peptides, etc., can solve the problems of increasing off-target, increasing the complexity of experimental operations, limiting applications, etc., and achieves wide application prospects, transcription activation The effect of improving efficiency and lowering technical threshold

Active Publication Date: 2018-02-23
SUN YAT SEN UNIV
View PDF3 Cites 15 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • High-efficiency artificial activating transcription factor dCas9-TV, and coding gene and applications thereof
  • High-efficiency artificial activating transcription factor dCas9-TV, and coding gene and applications thereof
  • High-efficiency artificial activating transcription factor dCas9-TV, and coding gene and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Construction of CRISPR / Cas9-derived artificial transcriptional activators

[0046] The codons of the 10th amino acid "D" and the 840th amino acid "H" of pcoCas9 (plant codon-optimized SpCas9) were replaced with the codons of amino acid "A" by PCR site-directed mutagenesis technology to obtain nuclease inactive Cas9, namely dCas9. The primers used are as follows:

[0047] Cas9-D10A-F: GTACTCTATCGGACTTGCTATCGGAACCAACTCTG

[0048] Cas9-D10A-R: CAGAGTTGGTTCCGATAGCAAGTCCGATAGAGTAC

[0049] Cas9-H840A-F: TGATTACGATGTTGATGCTATCGTTCCACAGTCTT

[0050] Cas9-H840A-R: AAGACTGTGGAACGATAGCATCAACATCGTAATCA

[0051] Q5 high-fidelity PCR polymerase was purchased from NEB Company, and the site-directed mutagenesis PCR reaction system was as follows:

[0052]

[0053] The HBT-pcoCas9 plasmid was constructed and preserved by our laboratory. Reaction program: denaturation at 98°C for 30 s; denaturation at 98°C for 10 s, annealing at 55°C for 30 s, extension at 72°C for 10 min, 16...

Embodiment 2

[0062] Transcriptional activation of Arabidopsis WRKY30 gene based on artificial transcriptional activator dCas9-TV

[0063] (1) Design of Arabidopsis WRKY30 gene promoter target sequence and construction of gRNA expression vector

[0064] The promoter sequence of the Arabidopsis WRKY30 gene was searched from the NCBI database, and the target sequence "AAGAACGAAGAAAGCTGATGTGG" (the underlined PAM structure) was selected within 200 bp upstream of the transcription start site, and gRNA-WRKY30 was designed accordingly. For this gene, the present invention uses two expression cassettes AtU6-1:gRNA:TTTTTT and AtU6-26:gRNA:TTTTTT, both of which are constructed using the overlapping PCR method, and the overlapping sequence is a 20nt target sequence. AtU6-1 and AtU6-26 promoters have been cloned by our laboratory before. The PCR primers used to construct AtU6-1:gRNA:TTTTTT are as follows:

[0065] U6-1-SacI-F: CGAGAGCTCAGAAATCTCAAAATTCCG

[0066]U6-1-WRKY30-R: CATCAGCTTTCTTCGTTTC...

Embodiment 3

[0098] Transcriptional activation of Arabidopsis RLP23 gene based on artificial transcriptional activator dCas9-TV

[0099] (1) Design of Arabidopsis RLP23 gene promoter target sequence and construction of gRNA expression vector

[0100] Find the promoter sequence of the Arabidopsis RLP23 gene from the NCBI database, and select the target sequence "AAATCCCTTAACGTATAACT" within 200 bp upstream of the transcription start site TGG " (the underline is the PAM structure), and gRNA-RLP23 was designed accordingly. The AtU6-26:gRNA:TTTTTT expression cassette was constructed using the overlapping PCR method, and the overlapping sequence was the 20nt target sequence. The PCR primers used were as follows:

[0101] U6-26-SacI-F: CGAGAGCTCAGCTTTTTTTCTTCTTCT

[0102] U6-26-RLP23-R: AGTTATACGTTAAGGGATTTCAATCACTACTTCGACTCT

[0103] gRNA-RLP23-F:AAATCCCTTAACGTATAACTGTTTTAGAGCTAGAAATA

[0104] gRNA-SacI-R: CGAGAGCTCAAAAAAGCACCGACTCGGTGC

[0105] After the overlapping PCR final products w...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

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...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N9/22C12N15/62C12N15/82C12N15/113
CPCC07K2319/71C12N9/22C12N15/113C12N15/8213C12N2310/10
Inventor 黎镇祥李剑峰张丹丹熊翔宇
Owner SUN YAT SEN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap