Detailed analysis of targeted gene mutations caused by the Platinum-Fungal TALENs in Aspergillus oryzae RIB40 strain and a ligD disruptant
Detailed analysis of targeted gene mutations caused by the Platinum-Fungal TALENs in Aspergillus oryzae RIB40 strain and a ligD disruptant
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DOI：
10.1016/j.jbiosc.2016.09.014
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发表时间：
2017-03-01
影响因子：
2.8
通讯作者：
Yamada, Osamu
中科院分区：
文献类型：
--
作者：
Mizutani, Osamu;Arazoe, Takayuki;Yamada, Osamu
转录激活子样效应子核酸酶(TALENS)可以在特定的基因组位点产生DNA双链断裂，在许多生物体中被用作基因组编辑的工具。然而，在包括米曲霉在内的曲霉中，TALENS的使用尚未得到验证。在本研究中，我们通过瞬时表达高效白金真菌TALEN5(PtFg TALENS)，通过非同源末端连接错误修复(NHEJ)对米曲霉野生型菌株进行了基因组编辑。靶向突变被观察到为各种突变模式。特别是，大约一半的PtFg TALEN介导的缺失突变体在TALEN靶向区的缺失大于1kb。我们还对缺失NHEJ修复最后一步所涉及的LigD基因的米曲霉LigD干扰物(Delta LigD)进行了基于PtFg TALEN的基因组编辑，发现仍然获得了与野生型一样的突变。在这种情况下，与野生型中基于PtFg TALEN的基因组编辑相比，大片段缺失的比例降低了。总之，我们证明了PtFg TALEN具有足够的功能，可以通过NHEJ在稻曲霉中的错误而导致基因组编辑。此外，我们还发现，在米曲霉中使用TALENS进行基因组编辑往往会导致目标区域的大量缺失，而这一缺失会被LigD的缺失部分抑制。(C)2016年，日本生物技术学会。版权所有。
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. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.