Mizutani Osamu, Arazoe Takayuki, Toshida Kenji, Hayashi Risa, Ohsato Shuichi, Sakuma Tetsushi, Yamamoto Takashi, Kuwata Shigeru, Yamada Osamu
National Research Institute of Brewing, 3-7-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-0046, Japan.
Graduate School of Agriculture, Meiji University, 1-1-1 Higashi-Mita, Tama-ku, Kawasaki, Kanagawa 214-8571, Japan.
J Biosci Bioeng. 2017 Mar;123(3):287-293. doi: 10.1016/j.jbiosc.2016.09.014. Epub 2016 Oct 22.
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.
转录激活样效应核酸酶（TALENs）能够在目标基因组位点的特定位置产生DNA双链断裂，已在许多生物体中用作基因组编辑工具。然而，在包括米曲霉在内的曲霉菌中，TALENs的应用尚未得到验证。在本研究中，我们通过瞬时表达高效铂-真菌TALENs（PtFg TALENs），利用非同源末端连接（NHEJ）修复错误对米曲霉野生型菌株进行了基因组编辑。观察到靶向突变呈现出各种突变模式。特别是，大约一半的PtFg TALEN介导的缺失突变体在TALEN靶向区域有大于1 kb的缺失。我们还在缺乏参与NHEJ修复最后一步的ligD基因的米曲霉ligD破坏株（ΔligD）中进行了基于PtFg TALEN的基因组编辑，发现与野生型一样仍能获得突变。在这种情况下，与野生型中基于PtFg TALEN的基因组编辑相比，大缺失的比例降低了。总之，我们证明PtFg TALENs具有足够的功能，能够通过米曲霉中NHEJ修复错误来实现基因组编辑。此外，我们发现米曲霉中使用TALENs进行基因组编辑往往会在目标区域导致大的缺失，而ligD的缺失可部分抑制这种情况。
