Nishizawa-Yokoi Ayako, Cermak Tomas, Hoshino Tomoki, Sugimoto Kazuhiko, Saika Hiroaki, Mori Akiko, Osakabe Keishi, Hamada Masao, Katayose Yuichi, Starker Colby, Voytas Daniel F, Toki Seiichi
Plant Genome Engineering Research Unit (A.N.-Y., H.S., A.M., S.T.), Rice Applied Genomics Research Unit (T.H., K.S.), and Advanced Genomics Laboratory (M.H., Y.K.), National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan;Department of Genetics, Cell Biology, and Development and Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota 55455 (T.C., C.S., D.F.V.);Center for Collaboration among Agriculture, Industry, and Commerce, University of Tokushima, Tokushima 770-8503, Japan (K.O.); andKihara Institute for Biological Research, Yokohama City University, Yokohama 244-0813, Japan (S.T.).
Plant Genome Engineering Research Unit (A.N.-Y., H.S., A.M., S.T.), Rice Applied Genomics Research Unit (T.H., K.S.), and Advanced Genomics Laboratory (M.H., Y.K.), National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan;Department of Genetics, Cell Biology, and Development and Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota 55455 (T.C., C.S., D.F.V.);Center for Collaboration among Agriculture, Industry, and Commerce, University of Tokushima, Tokushima 770-8503, Japan (K.O.); andKihara Institute for Biological Research, Yokohama City University, Yokohama 244-0813, Japan (S.T.)
Plant Physiol. 2016 Feb;170(2):653-66. doi: 10.1104/pp.15.01542. Epub 2015 Dec 14.
We have established methods for site-directed mutagenesis via transcription activator-like effector nucleases (TALENs) in the endogenous rice (Oryza sativa) waxy gene and demonstrated stable inheritance of TALEN-induced somatic mutations to the progeny. To analyze the role of classical nonhomologous end joining (cNHEJ) and alternative nonhomologous end joining (altNHEJ) pathways in TALEN-induced mutagenesis in plant cells, we investigated whether a lack of DNA Ligase4 (Lig4) affects the kinetics of TALEN-induced double-strand break repair in rice cells. Deep-sequencing analysis revealed that the frequency of all types of mutations, namely deletion, insertion, combination of insertion with deletion, and substitution, in lig4 null mutant calli was higher than that in a lig4 heterozygous mutant or the wild type. In addition, the ratio of large deletions (greater than 10 bp) and deletions repaired by microhomology-mediated end joining (MMEJ) to total deletion mutations in lig4 null mutant calli was higher than that in the lig4 heterozygous mutant or wild type. Furthermore, almost all insertions (2 bp or greater) were shown to be processed via copy and paste of one or more regions around the TALENs cleavage site and rejoined via MMEJ regardless of genetic background. Taken together, our findings indicate that the dysfunction of cNHEJ leads to a shift in the repair pathway from cNHEJ to altNHEJ or synthesis-dependent strand annealing.
我们已经建立了通过转录激活样效应核酸酶（TALENs）对内源水稻（Oryza sativa）蜡质基因进行定点诱变的方法，并证明了TALEN诱导的体细胞突变能够稳定遗传给后代。为了分析经典非同源末端连接（cNHEJ）和替代非同源末端连接（altNHEJ）途径在植物细胞中TALEN诱导诱变中的作用，我们研究了DNA连接酶4（Lig4）的缺失是否会影响水稻细胞中TALEN诱导的双链断裂修复动力学。深度测序分析表明，lig4缺失突变体愈伤组织中所有类型突变（即缺失、插入、插入与缺失组合以及替换）的频率高于lig4杂合突变体或野生型。此外，lig4缺失突变体愈伤组织中大片段缺失（大于10 bp）和通过微同源性介导的末端连接（MMEJ）修复的缺失占总缺失突变的比例高于lig4杂合突变体或野生型。此外，几乎所有插入（2 bp或更大）都显示是通过TALEN切割位点周围一个或多个区域的复制和粘贴进行处理，并通过MMEJ重新连接，而与遗传背景无关。综上所述，我们的研究结果表明，cNHEJ功能障碍导致修复途径从cNHEJ转变为altNHEJ或合成依赖链退火。
