title: Detailed analysis of targeted gene mutations caused by the Platinum-Fungal TALENs in Aspergillus oryzae RIB40 strain and a ligD disruptant.
abstract: 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 TALENs (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 (ΔligD) 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.
authors: National Research Institute of Brewing, 3-7-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-0046, Japan. Electronic address: mizutani@nrib.go.jp.
authors: Osamu
authors: O
authors: Mizutani
authors: Graduate School of Agriculture, Meiji University, 1-1-1 Higashi-Mita, Tama-ku, Kawasaki, Kanagawa 214-8571, Japan.
authors: Takayuki
authors: T
authors: Arazoe
authors: National Research Institute of Brewing, 3-7-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-0046, Japan.
authors: Kenji
authors: K
authors: Toshida
authors: National Research Institute of Brewing, 3-7-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-0046, Japan.
authors: Risa
authors: R
authors: Hayashi
authors: Graduate School of Agriculture, Meiji University, 1-1-1 Higashi-Mita, Tama-ku, Kawasaki, Kanagawa 214-8571, Japan.
authors: Shuichi
authors: S
authors: Ohsato
authors: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan.
authors: Tetsushi
authors: T
authors: Sakuma
authors: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan.
authors: Takashi
authors: T
authors: Yamamoto
authors: Graduate School of Agriculture, Meiji University, 1-1-1 Higashi-Mita, Tama-ku, Kawasaki, Kanagawa 214-8571, Japan.
authors: Shigeru
authors: S
authors: Kuwata
authors: National Research Institute of Brewing, 3-7-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-0046, Japan.
authors: Osamu
authors: O
authors: Yamada
mesh_terms: D001236:Aspergillus oryzae / Q000145:classification / Q000235:genetics*; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics*; D017353:Gene Deletion; D000072669:Gene Editing / Q000379:methods*; D018390:Gene Targeting; D016681:Genome, Fungal / Q000235:genetics; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation / Q000235:genetics*; D010984:Platinum / Q000378:metabolism*; D000069896:Transcription Activator-Like Effector Nucleases / Q000378:metabolism*
chemical_list: D005656:Fungal Proteins; D010984:Platinum; D000069896:Transcription Activator-Like Effector Nucleases
keywords: Aspergillus oryzae; Genome editing; Large deletion; Nonhomologous end-joining; Transcription activator-like effector nucleases; ligD