Zhu Min, Yan Lang, Zhan Zhitian, Chen Hong, Wang Dantong, Xu Meilian, Zheng Zhenping, Zhang Yujie, Yang Ning, Wu Junhua, Zhan Huadong, Tian Yanan, Xiong Lizhong, He Yubing
National Key Laboratory of Crop Genetic Improvement and National Centre of Plant Gene Research (Wuhan), Hubei Hongshan Laboratory, Huazhong Agricultural University, Wuhan, China.
State Key Laboratory of Crop Gene Resources and Breeding, Institute of Crop Sciences (ICS), Chinese Academy of Agricultural Sciences (CAAS), Beijing, China.
Plant Biotechnol J. 2025 Jul 13. doi: 10.1111/pbi.70257.
A critical step in generating gene-edited plants is the removal of CRISPR-related transgenes from T plants and their progenies, a process that is generally time-consuming and labour-intensive. We previously reported a Transgene Killer CRISPR (TKC) technology that enables self-elimination of transgenes after the targeted gene has been edited. However, we observed that a small number of T plants generated by TKC still retained the transgenes. Herein, we first integrated Cas9 and guide RNA (gRNA) with the RUBY reporter gene (RUBY-CRISPR) to monitor the Cas9/sgRNA expression and track the presence or absence of transgenes in the T generation and its progenies. We then combined the RUBY-CRISPR unit with several TKC variants to develop four RUBY-TKC (TKC2) systems including TKC2.1, TKC2.2, TKC2.3 and TKC2.4, to facilitate the elimination of escaped transgene plants. Compared to non-TKC, TKC alone and RUBY-CRISPR, our TKC2s were much more efficient in the generation of transgene-free edited progenies by up to 100% in the T generation. TKC2s not only omit the need for screening of the plants with transgenes in the T generation, but also enable visualisation of the escape of plants with transgenes in the following progenies. The TKC2 systems developed here provide straightforward yet highly effective approaches for the generation of transgene-free edited plants for either rice functional genomics or genetic improvement, with potential applications in gene editing of other crops.
培育基因编辑植物的关键一步是从T代植株及其后代中去除与CRISPR相关的转基因，这一过程通常既耗时又费力。我们之前报道了一种转基因杀手CRISPR（TKC）技术，该技术能够在目标基因被编辑后实现转基因的自我消除。然而，我们观察到由TKC产生的少数T代植株仍保留着转基因。在此，我们首先将Cas9和引导RNA（gRNA）与红宝石报告基因（红宝石-CRISPR）整合，以监测Cas9/sgRNA的表达，并追踪T代及其后代中转基因的存在与否。然后，我们将红宝石-CRISPR单元与几种TKC变体相结合，开发了四个红宝石-TKC（TKC2）系统，包括TKC2.1、TKC2.2、TKC2.3和TKC2.4，以促进对逃逸转基因植株的消除。与非TKC、单独的TKC和红宝石-CRISPR相比，我们的TKC2在产生无转基因编辑后代方面效率更高，在T代中高达100%。TKC2不仅无需在T代中筛选带有转基因的植株，还能在后续后代中可视化带有转基因植株的逃逸情况。这里开发的TKC2系统为培育用于水稻功能基因组学或遗传改良的无转基因编辑植物提供了直接而高效的方法，在其他作物的基因编辑中具有潜在应用价值。
