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<p>Genome editing with CRISPR/Cas9 enables precise DNA modifications in plant genomes but raises biosecurity and safety concerns due to potential off-target effects. Developing detection methods is crucial. We developed a cost-effective, low-technical loop-mediated isothermal amplification (LAMP) system to detect the Cas9 cassette at the DNA level in genome-edited plants. This method rapidly amplifies DNA in 30&#xa0;min, with visual detection facilitated by hydroxynaphthol blue (HNB), changing color from violet to sky blue. Our optimized LAMP-HNB reaction is sensitive enough to detect as few as one copy of the target DNA. To our knowledge, this novel assay is the first isothermal nucleic acid amplification method targeting the Cas9 cassette in genome-edited plants, offering LAMP-HNB as a valuable tool for diagnostic and monitoring tests. It is particularly useful for laboratories prioritizing safety and accuracy in detecting genome-edited modifications in agricultural products, highlighting the importance of addressing ethical and safety implications in genome editing.</p>

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Rapid and Specific Colorimetric Assay for Detecting CRISPR-Cas9 in Genome-Edited Plants Using Loop-Mediated Isothermal Amplification

  • 期刊论文

  • 发表日期：

    2025年1月17日

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  • Alberto Frango,
  • Qiming Chen,
  • Hua Zhang,
  • Zhanmin Liu

摘要

Genome editing with CRISPR/Cas9 enables precise DNA modifications in plant genomes but raises biosecurity and safety concerns due to potential off-target effects. Developing detection methods is crucial. We developed a cost-effective, low-technical loop-mediated isothermal amplification (LAMP) system to detect the Cas9 cassette at the DNA level in genome-edited plants. This method rapidly amplifies DNA in 30 min, with visual detection facilitated by hydroxynaphthol blue (HNB), changing color from violet to sky blue. Our optimized LAMP-HNB reaction is sensitive enough to detect as few as one copy of the target DNA. To our knowledge, this novel assay is the first isothermal nucleic acid amplification method targeting the Cas9 cassette in genome-edited plants, offering LAMP-HNB as a valuable tool for diagnostic and monitoring tests. It is particularly useful for laboratories prioritizing safety and accuracy in detecting genome-edited modifications in agricultural products, highlighting the importance of addressing ethical and safety implications in genome editing.