模块化组装的多重 Prime 编辑器，用于同时编辑水稻中具有重要农艺学意义的基因。
Modularly assembled multiplex prime editors for simultaneous editing of agronomically important genes in rice.
机构信息
Division of Plant Science and Technology, Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA.
State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China; Center for Crop Functional Genomics and Molecular Breeding, China Agricultural University, Beijing 100193, China.
出版信息
Plant Commun. 2024 Feb 12;5(2):100741. doi: 10.1016/j.xplc.2023.100741. Epub 2023 Oct 26.
Prime editing (PE) technology enables precise alterations in the genetic code of a genome of interest. PE offers great potential for identifying major agronomically important genes in plants and editing them into superior variants, ideally targeting multiple loci simultaneously to realize the collective effects of the edits. Here, we report the development of a modular assembly-based multiplex PE system in rice and demonstrate its efficacy in editing up to four genes in a single transformation experiment. The duplex PE (DPE) system achieved a co-editing efficiency of 46.1% in the T generation, converting TFIIAγ5 to xa5 and xa23 to Xa23. The resulting double-mutant lines exhibited robust broad-spectrum resistance against multiple Xanthomonas oryzae pathovar oryzae (Xoo) strains in the T generation. In addition, we successfully edited OsEPSPS1 to an herbicide-tolerant variant and OsSWEET11a to a Xoo-resistant allele, achieving a co-editing rate of 57.14%. Furthermore, with the quadruple PE (QPE) system, we edited four genes-two for herbicide tolerance (OsEPSPS1 and OsALS1) and two for Xoo resistance (TFIIAγ5 and OsSWEET11a)-using one construct, with a co-editing efficiency of 43.5% for all four genes in the T generation. We performed multiplex PE using five more constructs, including two for triplex PE (TPE) and three for QPE, each targeting a different set of genes. The editing rates were dependent on the activity of pegRNA and/or ngRNA. For instance, optimization of ngRNA increased the PE rates for one of the targets (OsSPL13) from 0% to 30% but did not improve editing at another target (OsGS2). Overall, our modular assembly-based system yielded high PE rates and streamlined the cloning of PE reagents, making it feasible for more labs to utilize PE for their editing experiments. These findings have significant implications for advancing gene editing techniques in plants and may pave the way for future agricultural applications.
碱基编辑（PE）技术能够精确地改变感兴趣的基因组中的遗传密码。PE 具有很大的潜力，可以识别植物中重要的农艺性状基因，并将其编辑为优良的变体，理想情况下同时靶向多个基因座，以实现编辑的综合效应。在这里，我们报告了在水稻中开发基于模块化组装的多重 PE 系统，并证明了其在单个转化实验中编辑多达四个基因的功效。双碱基编辑（DPE）系统在 T 代中实现了 46.1%的共编辑效率，将 TFIIAγ5 转化为 xa5，将 xa23 转化为 Xa23。在 T 代中，产生的双突变系表现出对多种水稻白叶枯病菌（Xoo）菌株的广谱抗性。此外，我们成功地将 OsEPSPS1 编辑为抗除草剂变体，将 OsSWEET11a 编辑为抗 Xoo 等位基因，共编辑率为 57.14%。此外，使用四重碱基编辑（QPE）系统，我们使用一个构建体编辑了四个基因——两个用于除草剂耐受性（OsEPSPS1 和 OsALS1），两个用于抗 Xoo（TFIIAγ5 和 OsSWEET11a），在 T 代中所有四个基因的共编辑效率为 43.5%。我们使用五个更多的构建体进行了多重 PE，包括两个用于三重碱基编辑（TPE）和三个用于 QPE，每个构建体针对不同的基因集。编辑率取决于 pegRNA 和/或 ngRNA 的活性。例如，ngRNA 的优化将一个靶基因（OsSPL13）的 PE 率从 0%提高到 30%，但对另一个靶基因（OsGS2）的编辑没有改善。总的来说，我们基于模块化组装的系统产生了高的 PE 率，并简化了 PE 试剂的克隆，使得更多的实验室能够将 PE 用于他们的编辑实验。这些发现对推进植物中的基因编辑技术具有重要意义，并可能为未来的农业应用铺平道路。
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