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成果報告書詳細
管理番号20170000000152
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 進化工学的および分子動力学的手法による新規ゲノム編集システムの創出
公開日2017/4/28
報告書年度2016 - 2016
委託先名国立大学法人徳島大学 学校法人明治大学 国立研究開発法人理化学研究所
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Genome editing tools, such as ZFNs, TALENs, and CRISPR/Cas9 were currently well-established as efficient biotechnological tools as rapid and reliable target-specific mutagenesis systems. However, the patents for these technologies have been issued by researchers in Europe and the United states, and therefore, it has been strongly desired to develop the novel genome editing tools that contribute to activation of Japanese biotechnological industry. Our aim is to develop the novel genome editing systems from unutilized molecular tools.
(1) Development of a novel genome editing tool by the study of molecular evolution engineering
We found a novel genome editing system, which has not been utilized for genome editing, from a photosynthetic bacteria, and it named this system as "TiD". The domain of TiD system for the recognition of target sequences was determined by the bacterial-based screening system. Based on the determined recognition affinity to the target sequences, the model target loci on benthamiana tobacco genome or tomato genome were designed. Using the expression system of the TiD system for plants, the target-specific mutation was introduced on the target sequences in benthamiana tobacco and tomato genomes.
(2) Searching for novel CRISPR alleles by in silico analysis
To identify unidentified TiD systems in microbial genome DNA sequences, a pipeline containing several programs for sequence similarity evaluation, functional analysis, and operon analysis was constructed. After retrieved the candidate sequences, the several unidentified TiD systems were identified by using the developed pipeline.
(3) Development of a novel genome editing system by structural and molecular dynamics analyses approaches
Molecular dynamics analysis was performed using the Cas9-RNA-DNA complex as a model genome editing tool of protein-nucleotides complex. Based on the analysis, we found that the mobility of the nuclease domain within the complex might be effective for DNA cleavage activity by the Cas9 nuclease domain.
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