Detailed analysis of targeted gene mutations caused by the Platinum-Fungal TALENs in Aspergillus oryzae RIB40 strain and a ligD disruptant

作者：Mizutani Osamu^*; Arazoe Takayuki; Toshida Kenji; Hayashi Risa; Ohsato Shuichi; Sakuma Tetsushi; Yamamoto Takashi; Kuwata Shigeru; Yamada Osamu

来源：Journal of Bioscience and Bioengineering, 2017, 123(3): 287-293.

DOI：10.1016/j.jbiosc.2016.09.014

• Aspergillus oryzae
• Genome editing
• Transcription activator-like effector nucleases
• ligD
• Large deletion
• Nonhomologous end-joining

Transcription activator-like effector nucleases (TALENs), which can generate DNA double-strand breaks at specific sites in the desired genome locus, have been used in many organisms as a tool for genome editing. In Aspergilli, including Aspergillus oryzae, however, the use of TALENs has not been validated. In this study, we performed genome editing of A. oryzae wild-type strain via error of nonhomologous end joining (NHEJ) repair by transient expression of high-efficiency Platinum-Fungal TALEN5 (PtFg TALENs). Targeted mutations were observed as various mutation patterns. In particular, approximately half of the PtFg TALEN-mediated deletion mutants had deletions larger than 1 kb in the TALEN-targeting region. We also conducted PtFg TALEN-based genome editing in A. oryzae ligD disruptant (Delta ligD) lacking the ligD gene involved in the final step of the NHEJ repair and found that mutations were still obtained as well as wild-type. In this case, the ratio of the large deletions reduced compared to PtFg TALEN-based genome editing in the wild-type. In conclusion, we demonstrate that PtFg TALENs are sufficiently functional to cause genome editing via error of NHEJ in A. oryzae. In addition, we reveal that genome editing using TALENs in A. oryzae tends to cause large deletions at the target region, which were partly suppressed by deletion of ligD.

• 出版日期2017-3