Elucidation of the mechanism for large deletions in Aspergillus oryzae caused by genome editing via error of non-homologous end-joining repair.

2018 Fiscal Year Final Research Report

• Project/Area Number: 16K07706
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Applied biochemistry
• Research Institution: University of the Ryukyus (2017-2018); National Research Institute of Brewing (2016)
• Principal Investigator: MIZUTANI Osamu 琉球大学, 農学部, 准教授 (10443996)
• Project Period (FY): 2016-04-01 – 2019-03-31
• Keywords: ゲノム編集 / 黄麹菌 / TALEN / CRISPR/Cas9 / 大規模欠失 / DNA 切断修復 / ligD

Outline of Final Research Achievements

We performed genome editing of Aspergillus oryzae wild-type strain via error of nonhomologous end-joining (NHEJ) repair by transient expression of TALENs. Targeted mutations were observed as various mutation patterns. Notably, about half of the TALEN-mediated deletion mutants had deletions larger than 1 kb in the TALEN-targeting region. In addition, when we performed genome editing using a ribonucleoprotein complex formed from Cas9 enzyme in A. oryzae wild-type strain, large deletions were also observed. Therefore, it was suggested that the large deletion was not depend on the cleavage styles of the genome editing tool. To examine the mechanisms for these large deletions and the influence of alternative NHEJ repairs, we conducted genome editing in A. oryzae ligD disruptant, which involved in the final step of NHEJ repair. As a result, the genome edited mutations were still observed as well as wild-type and the ratio of the large deletions reduced compared with that of wild-type strain.

Free Research Field

応用微生物学

Academic Significance and Societal Importance of the Research Achievements

本研究は、麹菌の新規育種を目指して、ゲノム編集ツールの一つである TALEN を用いた非相同末端結合修復エラーによるゲノム編集を行ったところ、酵母や高等動植物で見られるような数 bp の欠失の他に 1 kb 以上もの大規模な欠失が観察されたことに端を発している。本研究成果により、従来、他の生物と同様と考えられていた麹菌の DNA ２本鎖切断修復機構において、麹菌独自の機構が存在する可能性が示唆され、このメカニズムの解明により新規な麹菌育種法の開発等に繋がると期待される。