Abstract

Target activation-induced cytidine deaminase (Target-AID), a novel CRISPR/Cas9-based genome-editing tool, confers the base-editing capability on the Cas9 genome-editing system. It involves the fusion of cytidine deaminase (CDA), which catalyzes cytidine (C) to uridine (U) substitutions, to the mutated nickase-type nCas9 or deactivated-type dCas9. To confirm and extend the applicability of the Target-AID genome-editing system in tomatoes (Solanum lycopersicum L.), we transformed the model tomato cultivar “Micro-Tom” and commercial tomato cultivars using this system by targeting SlDELLA, which encodes a negative regulator of the plant phytohormone gibberellic acid (GA) signaling pathway. We confirmed that the nucleotide substitutions were induced by the Target-AID system, and we isolated mutants showing high GA sensitivity in both “Micro-Tom” and the commercial cultivars. Moreover, by successfully applying this system to ETHYLENE RECEPTOR 1 (SlETR1) with single sgRNA targeting, double sgRNA targeting, as well as dual-targeting of both SlETR1 and SlETR2 with a single sgRNA, we demonstrated that the Target-AID genome-editing system is a promising tool for molecular breeding in tomato crops. This study highlights an important aspect of the scientific and agricultural potential of the combinatorial use of the Target-AID and other base-editing systems.

中文翻译：

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Target-AID 对番茄育种性状和植物激素信号的修饰，基因组编辑系统可诱导有效的核苷酸替代

摘要

靶向激活诱导的胞苷脱氨酶 (Target-AID) 是一种基于 CRISPR/Cas9 的新型基因组编辑工具，赋予 Cas9 基因组编辑系统的碱基编辑能力。它涉及胞苷脱氨酶 (CDA) 与突变的切口酶型 nCas9 或失活型 dCas9 的融合，该酶催化胞苷 (C) 到尿苷 (U) 取代。为了确认和扩展 Target-AID 基因组编辑系统在番茄 ( Solanum lycopersicum L.) 中的适用性，我们通过靶向SlDELLA转化了模型番茄品种“Micro-Tom”和使用该系统的商业番茄品种，它编码植物激素赤霉酸（GA）信号通路的负调节剂。我们确认核苷酸取代是由 Target-AID 系统诱导的，我们分离出在“Micro-Tom”和商业栽培品种中均表现出高 GA 敏感性的突变体。此外，通过将该系统成功应用于乙烯受体 1 ( SlETR1 )，具有单 sgRNA 靶向、双 sgRNA 靶向以及SlETR1和SlETR2的双靶向使用单个 sgRNA，我们证明 Target-AID 基因组编辑系统是番茄作物分子育种的有前途的工具。这项研究突出了 Target-AID 和其他碱基编辑系统组合使用的科学和农业潜力的一个重要方面。