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成果報告書詳細
管理番号20170000000505
タイトル*平成28年度中間年報 植物等の生物を用いた高機能品生産技術の開発 植物の生産性制御に係る共通基盤技術開発 日本発新規ゲノム編集技術の研究開発
公開日2017/7/6
報告書年度2016 - 2016
委託先名国立大学法人筑波大学
プロジェクト番号P16009
部署名材料・ナノテクノロジー部
和文要約
英文要約Title:Summary for report 2016 Research & Development Program
Development of bio-production technologies of valuable substances using plants
-Development of basic and fundamental technologies for bio-production using plant
- Development of novel genome editing technology originated in Japan

i ) Development of genome editing technology by PODiR system
The development of genome editing technology, particularly the CRISPR/Cas9 system, is going to greatly change the future of the human life now, and that the technique has been taken not only in a medical field, but also in most industrial that used a creature. However, most patents of the genome editing technology, containing CRISPR/Cas9 system, are acquired monopolistically in foreign countries, give the Japanese bioindustries a big blow, and are going to invite a big loss of the national interest. When Maseda et al. analyzed the bacterial resistant acquisition mechanism to antibiotics, they found the self- genome editing mechanism that it was thought that a creature had in common. Therefore, based on this mechanism, we develop a new genome editing system (PODiR system) which is totally different from CRISPR/Cas9 and TALENT systems, establish Japan original genome editing technology enabling the modification at everywhere of genomes against all species, and aim at the establishment of the genome editing technology to take over them having the patent abroad.
 We tried to construct E. coli assay cells to measure exact efficiency of the genome editing in PODiR sequences. We introduced the drug-resistant marker which lost a function by introduction of the PODiR sequence in the E. coli orgami(DE3) stock, origami2(DE3) stock or NOVA Blue(DE3) stock under T7 promoter. It was thought that the transcription of this drug-resistant marker was promoted remarkably by addition of IPTG. Therefore we tried to measure amount of transcription and efficiency of the genome editing by adding various densities of IPTG. We tried to check to increase the efficiency of the self- genome editing, associated with an increase of the transcription.
ii ) Development of genome editing technology by PODiR system in various sequence (non-PODiR)
 The accurate self- genome editing that we aimed at with PODiR sequence happens in PODiR system, and doesn’t do in other parts. However, we clarified that only introduction of the mimic oligo nucleic acid in the PODiR system into a cell let organisms genome carry out genome editing in the all sequences. Therefore, we tried to construct E. coli assay cells to measure exact efficiency of the genome editing in various sequences (non-PODiR sequences) like i).
Results
i & ii ) We constructed the assay E. coli cells for assay of PODiR system,E. coli BL21(DE3) derivatives, E. coli BL21(DE3)-for PODiR and -for nonPODiR. When we added 1 mM IPTG in culture, the insertion in PODiR sequence deleted exactly and its resulting cell expressed Cm resistance by change from the dysfuncion Cm resistance gene to function one in E. coli BL21(DE3)-for PODiR cell, but not in 0 mM IPTG or in BL21(DE3)-for non-PODiR cell. These results suggested that the increase of transcription induced the deletion in PODiR system, and the efficiency of deletion in PODiR sequence increased only in increase of it. The assay for introducing the oligonucleotides is in progress.
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