Method for enhancing Cas9 and Cas9 derived protein mediated gene manipulation system and application

A protein and gene-derived technology, applied in the field of genetic engineering, to achieve the effect of good application value, strong gene activation ability, and good multi-gene activation ability

Active Publication Date: 2020-12-08
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the face of genes with high background expression (such as the Arabidopsis FLS2 gene), the dCas9-TV artificial transcription activator only weakly activates the expression of the target gene AtFLS2 by 1.6 times

Method used

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  • Method for enhancing Cas9 and Cas9 derived protein mediated gene manipulation system and application
  • Method for enhancing Cas9 and Cas9 derived protein mediated gene manipulation system and application
  • Method for enhancing Cas9 and Cas9 derived protein mediated gene manipulation system and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Example 1 Evaluating whether the artificial transcription activator dCas9-CCTV obtained by fusing the CC domain with dimerization activity to dCas9-TV undergoes protein dimerization in plant cells

[0086] A large number of natural transcription factors function in a dimerized form. In this example, the helical coiled CC domain (amino acid sequence shown in SEQ ID NO: 1) from the yeast transcriptional activator GCN4 was first introduced into the strong activator dCas9-TV (Li, Z .et al.A potent Cas9-derived gene activator for plant and mammalian cells.Nature plants, 3, 930–936, 2017), the specific test method is as follows:

[0087] 1. Construction of dCas9-CCTV transient expression vector

[0088] The HBT-dCas9-TV-FLAG vector (Li, Z. et al. A potent Cas9-derived gene activator for plant and mammalian cells. Nature plants, 3, 930–936, 2017.) was constructed and preserved by our laboratory, in which dCas9- The amino acid sequence of TV is shown in SEQ NO:2, and its codi...

Embodiment 2

[0136] Example 2 Evaluation of the activation effect of dCas9-CCTV in rice protoplasts

[0137] Using the same sgRNA in rice protoplasts, the gene activation effects of dimerization-capable dCas9-CCTV and original dCas9-TV were compared back-to-back ( figure 2 A). The specific test method is as follows:

[0138] 1. Construction of sgRNA transient expression vector

[0139] Refer to the methods described in published papers (Li, JF. et al. Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotianabenthamiana using guide RNA and Cas9. Nature biotechnology, 31, 688-69, 2013; Li, Z. et al. A potent Cas9-derived gene activator for plant and mammalian cells. Nature plants, 3, 930–936, 2017; Li, Z. et al. Targeted Transcriptional Activation in Plants Using a Potent Dead Cas9-Derived Synthetic GeneActivator. Current protocols in molecular biology, 127, e89, 2019), and construct an expression vector based on pUC119-OsU6apro-sgRNA (Li, Z. et al. 2019...

Embodiment 3

[0158] Example 3 Evaluating the effect of the dCas9-CCTV gene activation system on activating multiple genes in transgenic rice

[0159] A binary vector (dCas9-CCTV-PTGs-OsGW7-OsER1) was constructed by combining dCas9-CCTV with two sgRNAs, OsGW7-sgRNA and OsER1-sgRNA, where dCas9-CCTV uses the AtUBQ10 promoter and the two sgRNAs pass the ZmUBi-1 promoter The expression of the driven tRNA processing system (the nucleotide sequence of the binary vector is shown in SEQ NO: 11), and the rice transgene is carried out, and then RNA extraction and RT-qPCR experiments are performed on the 1-month-old transgenic rice leaves, the specific test method as follows:

[0160] 1. Construct the dCas9-CCTV gene activation system to co-target the stable transformation vector of OsGW7 and OsER1 and obtain gene activation and stable transformation of rice plants

[0161] The commercially synthesized dCas9-CCTV expression frame AtUBQ10pro-dCas9-CCTV-NOSterm and the commercially synthesized (Sibio)...

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Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a method for enhancing Cas9 and Cas9 derived protein mediated gene manipulation system and application. The method for enhancing a Cas9-derived artificial transcription factor is characterized in that polypeptide with a dimerization function is fused with Cas9 or a Cas9-derived protein. According to the above method, a CC structural domain with dimerization activity and an artificial activator dCas9-TV are fused to obtain a novel artificial activator dCas9-CCTV, the dCas9-CCTV is subjected to proteindimerization, and meanwhile, the dCas9-CCTV has stronger gene activation capability than the original dCas9-TV, has good polygene activation capability in a transgenic plant, and can be applied to enhancing the Cas9 and Cas9 derived protein mediated gene manipulation system, so that the system has better application value in the aspect of gene activated organism generation.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to a method for enhancing gene editing or expression regulation mediated by Cas9 and its derivative proteins, in particular to a method and application for enhancing a gene manipulation system mediated by Cas9 and its derivative proteins. Background technique [0002] In recent years, gene editing technology based on the CRISPR / Cas9 system has been widely used in the fields of biological gene function research, human clinical disease treatment, and crop genetic improvement due to its simplicity and high efficiency. The system mainly consists of nuclease Cas9 and guide RNA (sgRNA). Under the guidance of sgRNA, Cas9 binds to the target gene site, performs the function of nuclease, generates DNA double-strand breaks, and then causes mutations by the cell's endogenous repair mechanism. Researchers have found that when the nuclease activity of Cas9 is partially or completely l...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/22C12N15/113C12N15/82A01H5/00A01H6/20A01H6/46
CPCC12N9/22C12N15/113C12N15/8218C12N15/8261C12N2310/20
Inventor 李剑峰熊翔宇梁洁坪黎镇祥龚本强
Owner SUN YAT SEN UNIV
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