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<p>The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated protein (Cas)9 genome-editing technology has become a cornerstone for generating knockout mutations in plant functional genomics. To obtain genetically stable CRISPR-edited plants, the removal of exogenous CRISPR constructs through genetic segregation is imperative. However, current transgene-free strategies lack universality and operational simplicity. Here, we developed a modular CRISPR toolkit integrated with the widely applicable visual RUBY marker. This system achieved 100% editing efficiency in three independent CRISPR-Cas9 editing events in rice (cv. Zhonghua 11), enabled rapid visual identification of transgene-free progeny, and may provide a framework for future adaptation of CRISPR vectors to other plant species. Our design significantly accelerates the identification of edited lines while bypassing laborious molecular validation steps.</p>

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Seeing Red, Selecting True: RUBY-Reported Seed Marker Streamlines CRISPR-Clean Rice Breeding

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2025年10月3日

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  • Jin-Lei Liu,
  • Tao Yang,
  • Yu-Wei Fu,
  • Zhitian Zhan,
  • Hong Chen,
  • Han Cheng,
  • Jiankun Zhou,
  • Yi-Ming Wang,
  • Li-Jun Tang,
  • Wen-Qiang Chen,
  • Ming-Wei Wu,
  • Dake Zhao,
  • Chun-Ming Liu,
  • Yubing He,
  • Jinxin Liu

摘要

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated protein (Cas)9 genome-editing technology has become a cornerstone for generating knockout mutations in plant functional genomics. To obtain genetically stable CRISPR-edited plants, the removal of exogenous CRISPR constructs through genetic segregation is imperative. However, current transgene-free strategies lack universality and operational simplicity. Here, we developed a modular CRISPR toolkit integrated with the widely applicable visual RUBY marker. This system achieved 100% editing efficiency in three independent CRISPR-Cas9 editing events in rice (cv. Zhonghua 11), enabled rapid visual identification of transgene-free progeny, and may provide a framework for future adaptation of CRISPR vectors to other plant species. Our design significantly accelerates the identification of edited lines while bypassing laborious molecular validation steps.