将水稻内源 N-甲基嘌呤 DNA 糖基化酶与植物腺嘌呤碱基转换编辑器 ABE8e 融合,可在水稻植株中进行 A-K 碱基编辑

IF 4.6 4区 农林科学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY aBIOTECH Pub Date : 2024-03-21 DOI:10.1007/s42994-024-00138-8
Yucai Li, Shaoya Li, Chenfei Li, Chen Zhang, Lei Yan, Jingying Li, Yubing He, Yan Guo, Lanqin Xia
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引用次数: 0

摘要

在编辑窗口内同时进行腺嘌呤转换和反转的新型植物碱基编辑工程将极大地扩展碱基编辑在定向进化和作物改良中的应用范围和潜力。在这里,我们分离了一种水稻内源次黄嘌呤切除蛋白--N-甲基嘌呤DNA糖基化酶(OsMPG),并通过将OsMPG或其突变体mOsMPG与植物腺嘌呤转换碱基编辑器ABE8e融合,设计了两种植物A-to-K(K = G或T)碱基编辑器--rAKBE01和rAKBE02,用于在水稻中同时进行腺嘌呤转换和反转碱基编辑。我们进一步将 OsMPG 或 mOsMPG 与转录激活因子 VP64 结合,分别产生了 rAKBE03 和 rAKBE04。在水稻原生质体的五个内源位点上测试这四个 rAKBE,结果表明与 ABE8e 和 ABE8e-VP64 相比,rAKBE03 和 rAKBE04 能够实现更高水平的 A-G 碱基转换。此外,与 rAKBE02 和 rAKBE03 相比,rAKBE01 只能在一个内源基因座上实现 A-C/T 编辑,而 rAKBE04 则能显著提高水稻原生质体中 A-C/T 碱基转换效率,分别高达 6.57 倍和 1.75 倍。此外,虽然 rAKBE01 和 rAKBE04 没有诱导出具有 A-C 转换的稳定品系,但 rAKBE04 可在所有五个目标基因座上同时实现 A-G 和 A-T 转换和反转碱基编辑,在水稻稳定品系中的 A-G 转换和 A-T 反转编辑效率分别为 70.97% 至 92.31% 和 1.67% 至 4.84%。这些 rAKBEs 可提供不同的编辑产品组合,从而拓展了碱基编辑在作物改良中的多种应用前景。
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Fusion of a rice endogenous N-methylpurine DNA glycosylase to a plant adenine base transition editor ABE8e enables A-to-K base editing in rice plants

Engineering of a new type of plant base editor for simultaneous adenine transition and transversion within the editing window will greatly expand the scope and potential of base editing in directed evolution and crop improvement. Here, we isolated a rice endogenous hypoxanthine excision protein, N-methylpurine DNA glycosylase (OsMPG), and engineered two plant A-to-K (K = G or T) base editors, rAKBE01 and rAKBE02, for simultaneous adenine transition and transversion base editing in rice by fusing OsMPG or its mutant mOsMPG to a plant adenine transition base editor, ABE8e. We further coupled either OsMPG or mOsMPG with a transactivation factor VP64 to generate rAKBE03 and rAKBE04, respectively. Testing these four rAKBEs, at five endogenous loci in rice protoplasts, indicated that rAKBE03 and rAKBE04 enabled higher levels of A-to-G base transitions when compared to ABE8e and ABE8e-VP64. Furthermore, whereas rAKBE01 only enabled A-to-C/T editing at one endogenous locus, in comparison with rAKBE02 and rAKBE03, rAKBE04 could significantly improve the A-to-C/T base transversion efficiencies by up to 6.57- and 1.75-fold in the rice protoplasts, respectively. Moreover, although no stable lines with A-to-C transversion were induced by rAKBE01 and rAKBE04, rAKBE04 could enable simultaneous A-to-G and A-to-T transition and transversion base editing, at all the five target loci, with the efficiencies of A-to-G transition and A-to-T transversion editing ranging from 70.97 to 92.31% and 1.67 to 4.84% in rice stable lines, respectively. Together, these rAKBEs enable different portfolios of editing products and, thus, now expands the potential of base editing in diverse application scenario for crop improvement.

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