Kaya Hidetaka, Numa Hisataka, Nishizawa-Yokoi Ayako, Toki Seiichi, Habu Yoshiki
Institute of Agrobiological Sciences, National Agriculture and Food Research Organization (NARO) Tsukuba, Japan.
Advanced Analysis Center, National Agriculture and Food Research Organization (NARO) Tsukuba, Japan.
Front Plant Sci. 2017 Mar 13;8:302. doi: 10.3389/fpls.2017.00302. eCollection 2017.
Genome editing in plants becomes popular since the advent of sequence-specific nucleases (SSNs) that are simple to set up and efficient in various plant species. Although transcription activator-like effector nucleases (TALENs) are one of the most prevalent SSNs and have a potential to provide higher target specificity by their dimeric property, TALENs are sensitive to methylated cytosines that are present not only in transposons but also in active genes in plants. In mammalian cells, the methylation sensitivity of TALENs could be overcome by using a base-recognition module (N) that has a higher affinity to methylated cytosine. In contrast to mammals, plants carry DNA methylation at all cytosine contexts (CG, CHG, and CHH, where H represents A, C, or T) with various degrees and effectiveness of N module in genome editing in plants has not been explored. In this study, we designed sets of TALENs with or without N modules and examined their efficiency in genome editing of methylated regions in rice. Although improvement in genome editing efficiency was observed with N-TALENs designed to a stably methylated target, another target carrying cytosines with various levels of methylation showed resistance to both normal and N-TALENs. The results suggest that variability of cytosine methylation in target regions is an additional factor affecting the genome editing efficiency of TALENs.
自从序列特异性核酸酶（SSNs）出现以来，植物基因组编辑变得流行起来，这类酶易于操作且在各种植物物种中都很高效。尽管转录激活样效应因子核酸酶（TALENs）是最普遍的SSNs之一，并且因其二聚体特性有可能提供更高的靶标特异性，但TALENs对甲基化胞嘧啶敏感，甲基化胞嘧啶不仅存在于转座子中，也存在于植物的活性基因中。在哺乳动物细胞中，通过使用对甲基化胞嘧啶具有更高亲和力的碱基识别模块（N），可以克服TALENs的甲基化敏感性。与哺乳动物不同，植物在所有胞嘧啶背景（CG、CHG和CHH，其中H代表A、C或T）下都存在DNA甲基化，并且尚未探索N模块在植物基因组编辑中的不同程度和效果。在本研究中，我们设计了带有或不带有N模块的TALENs组，并检测了它们在水稻甲基化区域基因组编辑中的效率。尽管观察到针对稳定甲基化靶标的N-TALENs在基因组编辑效率上有所提高，但另一个携带不同甲基化水平胞嘧啶的靶标对正常TALENs和N-TALENs均表现出抗性。结果表明，靶标区域胞嘧啶甲基化的变异性是影响TALENs基因组编辑效率的另一个因素。
