植物细胞器，包括线粒体和叶绿体含有自己的基因组，它们分别编码许多对呼吸和光合作用必需的基因。 植物细胞器中的基因编辑是对植物遗传学和生物技术的未满足需求，因缺乏适当的工具来阻碍这些细胞器中的DNA。 在这项研究中，我们开发了由16种表达质粒组成的Golden Gate cloning 克隆系统1（8用于将所得蛋白质递送到线粒体，另外8用于递送至叶绿体）和424类转录激活效应因子阵列质粒，以组装DDDA - 定期的胞嘧啶碱基编辑器（DDCBE）2质粒，并使用所得的DDCBES在线粒体和叶绿体中有效地促进点诱变。
我们的DDCBES诱导莴苣或油菜籽愈伤组织碱基编辑，频率高达25％（线粒体）和38％（叶绿体）。 通过将DDCBE mRNA传递给莴苣原生质体，我们还显示出在叶绿体中进行的无DNA的基础编辑，以避免由DDCE编码质粒引起的偏离靶向突变。此外，我们通过在叶绿体16s rRNA基因中引入点突变，产生高达99％的莴苣愈伤组织和Plantlet，其高达99％，其抵抗链霉素或壮观霉素。
Plant organelles including mitochondria and chloroplasts contain their own genomes, which encode many genes essential for respiration and photosynthesis, respectively. Gene editing in plant organelles, an unmet need for plant genetics and biotechnology, has been hampered by the lack of appropriate tools for targeting DNA in these organelles. In this study, we developed a Golden Gate cloning system1, composed of 16 expression plasmids (8 for the delivery of the resulting protein to mitochondria and the other 8 for delivery to chloroplasts) and 424 transcription activator-like effector subarray plasmids, to assemble DddA-derived cytosine base editor (DdCBE)2 plasmids and used the resulting DdCBEs to efficiently promote point mutagenesis in mitochondria and chloroplasts. Our DdCBEs induced base editing in lettuce or rapeseed calli at frequencies of up to 25% (mitochondria) and 38% (chloroplasts). We also showed DNA-free base editing in chloroplasts by delivering DdCBE mRNA to lettuce protoplasts to avoid off-target mutations caused by DdCBE-encoding plasmids. Furthermore, we generated lettuce calli and plantlets with edit frequencies of up to 99%, which were resistant to streptomycin or spectinomycin, by introducing a point mutation in the chloroplast 16S rRNA gene.
Nature Plants, Published online: 01 July 2021;
doi:10.1038/s41477-021-00943-9
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