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植物中的叶绿体和线粒体 DNA 编辑。

Chloroplast and mitochondrial DNA editing in plants.

机构信息

Center for Genome Engineering, Institute for Basic Science, Daejeon, Republic of Korea.

Department of Chemistry, Seoul National University, Seoul, Republic of Korea.

出版信息

Nat Plants. 2021 Jul;7(7):899-905. doi: 10.1038/s41477-021-00943-9. Epub 2021 Jul 1.

Abstract

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 system, 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) 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.

摘要

植物细胞器(包括线粒体和叶绿体)含有各自的基因组,分别编码呼吸和光合作用所必需的许多基因。细胞器中的基因编辑是植物遗传学和生物技术的一项未满足的需求,但由于缺乏针对这些细胞器中 DNA 的适当工具,该技术受到了阻碍。在这项研究中,我们开发了一个 Golden Gate 克隆系统,由 16 个表达质粒(8 个用于将所得蛋白质递送到线粒体,另外 8 个用于递送到叶绿体)和 424 个转录激活因子样效应子亚阵列质粒组成,用于组装 DddA 衍生的胞嘧啶碱基编辑器(DdCBE)质粒,并使用所得的 DdCBE 在线粒体和叶绿体中高效地促进点突变。我们的 DdCBE 在生菜或油菜愈伤组织中的碱基编辑频率高达 25%(线粒体)和 38%(叶绿体)。我们还通过将 DdCBE mRNA 递送到生菜原生质体中来避免由 DdCBE 编码质粒引起的脱靶突变,从而在叶绿体中实现了无 DNA 的碱基编辑。此外,我们通过在叶绿体 16S rRNA 基因中引入点突变,产生了编辑频率高达 99%的生菜愈伤组织和植物,这些愈伤组织和植物对链霉素或壮观霉素具有抗性。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2604/8289734/2a01896258ca/41477_2021_943_Fig1_HTML.jpg

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