Table_1_In-Depth Characterization of greenflesh Tomato Mutants Obtained by CRISPR/Cas9 Editing: A Case Study With Implications for Breeding and Regulation.XLSX

Gene editing has already proved itself as an invaluable tool for the generation of mutants for crop breeding, yet its ultimate impact on agriculture will depend on how crops generated by gene editing technologies are regulated, and on our ability to characterize the impact of mutations on plant phenotype. A starting operational strategy for evaluating gene editing-based approaches to plant breeding might consist of assessing the effect of the induced mutations in a crop- and locus-specific manner: this involves the analysis of editing efficiency in different cultivars of a crop, the assessment of potential off-target mutations, and a phenotypic evaluation of edited lines carrying different mutated alleles. Here, we targeted the GREENFLESH (GF) locus in two tomato cultivars (‘MoneyMaker’ and ‘San Marzano’) and evaluated the efficiency, specificity and mutation patterns associated with CRISPR/Cas9 activity for this gene. The GF locus encodes a Mg-dechelatase responsible for initiating chlorophyll degradation; in gf mutants, ripe fruits accumulate both carotenoids and chlorophylls. Phenotypic evaluations were conducted on two transgene-free T2 ‘MoneyMaker’ gf lines with different mutant alleles (a small insertion of 1 nucleotide and a larger deletion of 123 bp). Both lines, in addition to reduced chlorophyll degradation, showed a notable increase in carotenoid and tocopherol levels during fruit ripening. Infection of gf leaves and fruits with Botrytis cinerea resulted in a significant reduction of infected area and pathogen proliferation compared to the wild type (WT). Our data indicates that the CRISPR/Cas9-mediated mutation of the GF locus in tomato is efficient, specific and reproducible and that the resulting phenotype is robust and consistent with previously characterized greenflesh mutants obtained with different breeding techniques, while also shedding light on novel traits such as vitamin E overaccumulation and pathogen resistance. This makes GF an appealing target for breeding tomato cultivars with improved features for cultivation, as well as consumer appreciation and health.

基因编辑（Gene editing）已被证实是作物育种突变体创制的宝贵工具，但其对农业的最终影响，将取决于基因编辑技术培育作物的监管方式，以及我们能否精准表征突变对植物表型的作用。用于评估基于基因编辑的作物育种策略的初始操作框架，可采用作物与位点特异性方式评估诱导突变的效应：具体包括分析某一作物不同栽培品种的编辑效率、评估潜在脱靶突变（off-target mutations），以及对携带不同突变等位基因的编辑株系开展表型鉴定。本研究以两个番茄栽培品种（‘MoneyMaker’与‘San Marzano’）中的GREENFLESH(GF)位点为靶向目标，评估了该基因相关CRISPR/Cas9的编辑效率、特异性与突变模式。GF位点编码一种负责启动叶绿素降解的镁脱螯合酶；在gf突变体中，成熟果实会同时积累类胡萝卜素与叶绿素。研究人员对两个携带不同突变等位基因的无转基因（transgene-free）T2代‘MoneyMaker’番茄gf株系开展了表型分析：其中一个株系携带1个核苷酸的小片段插入突变，另一个则携带123 bp的大片段缺失突变。与野生型（wild type, WT）相比，这两个株系不仅叶绿素降解能力显著减弱，果实成熟过程中的类胡萝卜素与生育酚（tocopherol，即维生素E）含量均有明显提升。用灰葡萄孢菌（Botrytis cinerea）侵染gf株系的叶片与果实后，其侵染面积与病原菌增殖量均较野生型显著降低。本研究数据表明，通过CRISPR/Cas9介导编辑番茄GF位点，具备高效、特异且可重复的特点，所得表型与此前通过其他育种技术获得的greenflesh突变体特征一致，同时还揭示了维生素E过量积累、病原菌抗性提升等全新性状。这使得GF位点成为培育具备更优栽培性状、消费者接受度与健康价值的番茄栽培品种的极具吸引力的育种靶点。