Method for increasing efficiency of homologous recombination-based gene editing in plant

A technology of gene editing and homologous recombination, applied in biochemical equipment and methods, other methods of inserting foreign genetic materials, genetic engineering, etc., can solve undisclosed problems

Active Publication Date: 2020-09-04
IND ACADEMIC COOP FOUND GYEONGSANG NAT UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] On the other hand, although Korean Laid-Open Patent No. 2017-0081268 discloses a single guide RNA (sgRNA) with a single guide RNA (sgRNA) that mediates sequence-specific cleavage for editing the target sequence of the tobacco rattle virus (TRV) sequence and the genome of a subject. The plant cell of the sequence structure and its gene editing application are related to the nucleic acid structure for editing the genome, but there is no disclosure of a method for improving the efficiency of gene editing based on homologous recombination in plants

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  • Method for increasing efficiency of homologous recombination-based gene editing in plant
  • Method for increasing efficiency of homologous recombination-based gene editing in plant
  • Method for increasing efficiency of homologous recombination-based gene editing in plant

Examples

Experimental program
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Effect test

Embodiment 1

[0102] Example 1. Homologous recombination efficiency analysis using anthocyanin labeling

[0103] To investigate the efficiency of homology-mediated double-strand DNA repair-based gene scissors in tomato crops, the 35S promoter was inserted as a transcription factor for regulation of anthocyanin synthesis by homology-mediated double-strand DNA repair. In the upstream promoter position of the transcription start position of the ANT1 gene, and through the overexpression of anthocyanins activated by the ANT1 gene to induce the formation of purple callus.

[0104] When the homology-mediated double-strand DNA repair template (sequence 29) can perform a homology-mediated double-strand DNA repair event by inserting pNos-NPTII-OCSt in the 35S promoter base sequence and its upstream, the acquisition card Namycin resistance. In this case, the upper base sequence having homology is 1043 bp, and the lower base sequence is 592 bp. And, two transcription activator-like effector nuclease ...

Embodiment 2

[0130] Example 2. Increased Homologous Recombination Frequency in Plants by SCR7 Pyrazine Treatment

[0131] Most DNA repair occurs through the non-homologous end-joining pathway, while repair based on homology-mediated double-strand DNA repair occurs in part. According to existing research reports, the efficiency of homology-mediated double-strand DNA repair can be improved in mammals by blocking the non-homologous end-joining pathway. SCR7 pyrazine, an inhibitor of mammalian ligase IV, was reported to increase homology-mediated double-strand DNA repair by approximately 19-fold in mice and 5-fold in human cell lines. According to the viewpoint proposed by Nishizawa-Yokoi et al. (2016, Plant Physiol.170(2):653-666), in plants, ligase IV can play an important role in the non-homologous end joining pathway of rice. However, there have been no reports of the effect of SCR7 pyrazines on homology-mediated double-strand DNA repair in plants. Therefore, the present inventors analyz...

Embodiment 3

[0144] Example 3. The kinetic model of the release of the bean yellow dwarf virus replication replicon

[0145] The inventors found the optimal time point for maximal release of the prototypic yellow dwarf virus replicon in the Agrobacterium-infected tomato cotyledon system. Viral replicons are released in prototypical form from linear triple-helical DNA delivered to the plant nucleus and amplified to hundreds to thousands per cell by rolling circle replication. Information on the kinetics of maximal replicon release can provide information on the optimal period for adapting a small molecule to tissue culture systems. To study the dynamics of replicon release, cotyledons were transformed with Agrobacterium containing the bean yellow dwarf virus vectors pLSL.GFP.R (Cermak et al., 2015), pLSL.R.GGFP and the non-viral vector pAGM4723. Virus prototypes were detected by PCR analysis using specific primers that amplify the junction of two large intergenic regions in genomic DNA iso...

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Abstract

The present invention relates to a method for increasing the efficiency of homologous recombination-based gene editing in a plant, comprising a step of optimizing temperature and photoperiod conditions, expressing factors necessary for homology-directed DNA repair (HDR) and factors for increasing the efficiency of HDR by using multiple replicons, or regulating an HDR pathway or a non-homologous end joining (NHEJ) pathway, during tissue culturing of plant cells.

Description

technical field [0001] The invention relates to a method for improving gene editing efficiency based on homologous recombination in plants. Background technique [0002] Genome correction technology through homologous recombination (HR, homologous recombination) or homology-mediated double-strand DNA repair (HDR, homology-directed DNA repair) in plant systems is considered a milestone in plant genetic engineering research field. Although homologous recombination often occurs during meiosis of haploid germ cells, it does not occur during mitosis of somatic cells. It is reported that homology-mediated double-strand DNA repair is a repair pathway that occurs together with non-homologous end joining (NHEJ) when DNA is damaged, and homology-mediated double-strand DNA repair occurs The frequency is 1 / 1000 of non-homologous end joining. According to reports in the early 1990s, double strand breaks (DSB, double strand break) in Nicotiana species can increase homology-mediated dou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/10C12N15/113C12N15/90C12N9/22
CPCC12N9/22C12N15/10C12N15/902C12N15/8213C12N15/8212C12N15/8216C12N15/8203C12N15/82C12N15/102C12N15/113C12N15/907C12N2310/10C12N2310/20
Inventor 金在演吴天万韦鲁·斯万卡雅尼米尔蒂·川金知谐成演瑀郑世正金贤晶
Owner IND ACADEMIC COOP FOUND GYEONGSANG NAT UNIV
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