Gene upregulation through genome editing is important for plant research and breeding. Targeted insertion of short transcriptional enhancers (STEs) into gene promoters may offer a universal solution akin to transgene-mediated overexpression while avoiding the drawbacks associated with transgenesis. Here, we introduce an “ activation” technique in rice that leverages well-characterized STEs for refined, heritable, and multiplexed gene upregulation. To address the scarcity of potent enhancers, we developed a large-scale mining approach and discovered a suite of STEs that are capable of enhancing gene expression in rice protoplasts. The integration of these STEs into eight rice genes resulted in substantial transcriptional upregulation in the edited plants, with up to 869.1-fold increases in their transcript levels. Employing a variety of STEs, we achieved delicate control of gene expression, enabling the fine-tuning of key phenotypic traits such as plant height. Our approach also enabled efficient multiplexed gene upregulation, with up to four genes activated simultaneously, significantly enhancing the nicotinamide mononucleotide metabolic pathway. Importantly, heritability studies from the T0 to T3 generations confirmed the stable and heritable nature of STE-driven gene activation. Collectively, our work demonstrates that coupled with STE mining, leveraging genome editing for activation and gene upregulation holds great promise to be widely adopted in fundamental plant research and crop breeding.

中文翻译：

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通过 CRISPR-Cas 介导的增强子敲入，实现植物中高效、多重基因上调


通过基因组编辑进行基因上调对于植物研究和育种非常重要。将短转录增强子（STE）定向插入基因启动子可能提供类似于转基因介导的过度表达的通用解决方案，同时避免与转基因相关的缺点。在这里，我们引入了一种水稻“激活”技术，该技术利用特征明确的 STE 进行精细、可遗传和多重的基因上调。为了解决强效增强子的稀缺问题，我们开发了一种大规模挖掘方法，并发现了一套能够增强水稻原生质体中基因表达的 STE。将这些 STE 整合到 8 个水稻基因中，导致编辑后的植物中转录水平大幅上调，其转录水平提高了 869.1 倍。通过采用各种 STE，我们实现了对基因表达的精细控制，从而能够微调株高等关键表型性状。我们的方法还实现了高效的多重基因上调，最多可同时激活四个基因，显着增强烟酰胺单核苷酸代谢途径。重要的是，从 T0 到 T3 代的遗传性研究证实了 STE 驱动的基因激活的稳定性和可遗传性。总的来说，我们的工作表明，与 STE 挖掘相结合，利用基因组编辑进行激活和基因上调有望在基础植物研究和作物育种中得到广泛采用。