Study on DNA repair mechanism via large-scale deletion in Aspergillus oryzae caused by genome editing due to error of non-homologous end-joining repair.

2022 Fiscal Year Final Research Report

• Project/Area Number: 19K05952
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 38060:Applied molecular and cellular biology-related
• Research Institution: University of the Ryukyus
• Principal Investigator: Mizutani Osamu 琉球大学, 農学部, 准教授 (10443996)
• Project Period (FY): 2019-04-01 – 2023-03-31
• Keywords: 麹菌 / ゲノム編集 / 人工ヌクレアーゼ (TALENs) / CRISPR/Cas9 / DNA 二本鎖切断修復

Outline of Final Research Achievements

We performed genome editing of Aspergillus oryzae wild-type strain via error of nonhomologous end-joining (NHEJ) repair by TALENs and CRISPR/Cas9. Targeted mutations were observed as various mutation patterns. Notably, about half of the mediated deletion mutants caused by genome editing had deletions larger than 1 kb in the targeting region of TALEN or CRISPR/Cas9.In this study, we aim to elucidate the mechanism of large deletions caused by double-strand breaks during genome editing in Aspergillus oryzae. To investigate whether this large deletion is related to the NHEJ pathway, we constructed disruptant of polD gene which may be related to the NHEJ pathway and performed genome editing using the strain as host. As a result, it was suggested that A. oryzae polD gene was not involved the large deletion via genome editing. On the other hand, genome analysis of a large deletion-suppressed mutant revealed 17 mutated genes.

Free Research Field

応用微生物学

Academic Significance and Societal Importance of the Research Achievements

本研究は、麹菌において非相同末端結合修復エラーを介したゲノム編集を行うと、酵母や高等動植物で観察されるような数 bp の欠失の他に 1 kb 以上もの大規模な欠失が高頻度で観察されることについて、どのような因子が大規模欠失に関与しているかを明らかにしようとしている。本研究が進展すれば、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、麹菌独自の機構の発見やこのメカニズムを利用した新たな麹菌育種法の開発等に繋がると期待される。