Modularly assembled multiplex prime editors for simultaneous editing of agronomically important genes in rice
Modularly assembled multiplex prime editors for simultaneous editing of agronomically important genes in rice
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DOI：
10.1016/j.xplc.2023.100741
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发表时间：
2023-10
影响因子：
10.5
通讯作者：
Ajay Gupta;Bo Liu;Saad Raza;Qi-Jun Chen;Bing Yang
中科院分区：
文献类型：
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作者：
Ajay Gupta;Bo Liu;Saad Raza;Qi-Jun Chen;Bing Yang
Prime 编辑 (PE) 技术可以精确改变目标基因组的遗传密码。 PE 在识别植物中具有重要农艺意义的基因并将其编辑成优良变体方面具有巨大潜力，理想情况下同时针对多个基因座以实现编辑的集体效应。在此，我们报告了水稻中基于模块化组装的多重 PE 系统的开发，并证明了其在单个转化实验中编辑多达四个基因的功效。双工PE（DPE）系统在T0代中实现了46.1%的共编辑效率，将TFIIAγ5toxa5andxa23转化为Xa23SW11。由此产生的双突变株系在 T1 代中对多种稻黄单胞菌 (Xanthomonas oryzaepathovaroryzae) (Xoo) 菌株表现出强大的广谱抗性。此外，我们成功地将OsEPSPS1编辑为耐除草剂变异体和OsSWEET11ato aXoo抗性等位基因，共编辑率达到57.14%。此外，利用四重 PE (QPE) 系统，我们使用一个构建体编辑了四个基因，其中两个用于除草剂耐受性（OsEPSPS1和OsALS1），两个用于抗性（TFIIAγ5和OsSWEET11a），T0代中所有四个基因的共同编辑效率为43.5%。我们使用另外五个构建体进行多重 PE，其中两个用于三重 PE (TPE)，三个用于 QPE，每个构建体针对一组不同的基因。编辑率取决于 pegRNA 和/或 ngRNA 的活性。例如，ngRNA 的优化将其中一个目标 (OsSPL13) 的 PE 率从 0% 提高到 30%，但没有改善另一个目标 (OsGS2) 的编辑。总体而言，我们基于模块化组装的系统产生了高 PE 率，并简化了 PE 试剂的克隆，使更多实验室可以利用 PE 进行编辑实验。这些发现对于推进植物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。
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 T0generation, convertingTFIIAγ5toxa5andxa23toXa23SW11. The resulting double-mutant lines exhibited robust broad-spectrum resistance against multipleXanthomonas oryzaepathovaroryzae(Xoo) strains in the T1generation. In addition, we successfully editedOsEPSPS1to an herbicide-tolerant variant andOsSWEET11ato aXoo-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 (OsEPSPS1andOsALS1) and two forXooresistance (TFIIAγ5andOsSWEET11a)—using one construct, with a co-editing efficiency of 43.5% for all four genes in the T0generation. 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.