DataSheet_1_Identification of Transgene-Free CRISPR-Edited Plants of Rice, Tomato, and Arabidopsis by Monitoring DsRED Fluorescence in Dry Seeds.pdf

Efficient elimination of the editing machinery remains a challenge in plant biotechnology after genome editing to minimize the probability of off-target mutations, but it is also important to deliver end users with edited plants free of foreign DNA. Using the modular cloning system Golden Braid, we have included a fluorescence-dependent transgene monitoring module to the genome-editing tool box. We have tested this approach in Solanum lycopersicum, Oryza sativa, and Arabidopsis thaliana. We demonstrate that DsRED fluorescence visualization works efficiently in dry seeds as marker for the detection of the transgene in the three species allowing an efficient method for selecting transgene-free dry seeds. In the first generation of DsRED-free CRISPR/Cas9 null segregants, we detected gene editing of selected targets including homozygous mutants for the plant species tested. We demonstrate that this strategy allows rapid selection of transgene-free homozygous edited crop plants in a single generation after in vitro transformation.

在基因组编辑完成后，高效清除编辑元件以降低脱靶突变概率仍是植物生物技术领域的一大挑战，同时为终端用户提供不含外源DNA的编辑植株也至关重要。本研究采用模块化克隆系统Golden Braid，将荧光依赖型转基因监测模块整合至基因组编辑工具包中。我们在番茄（Solanum lycopersicum）、水稻（Oryza sativa）及拟南芥（Arabidopsis thaliana）中对该方法进行了测试。研究证实，DsRED荧光可视化可高效作为标记物在三种受试物种的干燥种子中检测转基因，从而为筛选无转基因干燥种子提供了一种高效方法。在不含DsRED的CRISPR/Cas9无转基因分离株的第一代子代中，我们于受试植物中检测到了选定靶点的基因编辑事件，其中包含纯合突变体。本研究证实，该策略可在体外转化后的单一代次中，快速筛选获得不含转基因的纯合编辑作物植株。