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<p>DNA methylation at specific CpG sites serves as a critical biomarker for disease diagnosis, yet existing detection methods often lack single-site resolution, are susceptible to interference from adjacent CpGs, or involve complex and time-consuming processes. Here we introduce EPILIGHT, a rapid and ratiometric CRISPR-based strategy designed for precise quantification of methylation level at individual CpG sites while effectively minimizing interference from flanking CpG sites. The approach integrates ultrafast bisulfite conversion, recombinase polymerase amplification (RPA), and a dual-channel CRISPR/Cas12a detection system to independently recognize methylated (C) and unmethylated (T) sequences. By computing the ratio of methylation-derived signal to the total fluorescence signal via a standard curve, EPILIGHT ensures accurate and reproducible quantification independent of DNA input or reaction efficiency. The entire procedure is completed within 70 min. We validated EPILIGHT by targeting two closely spaced CpG sites in the IFI44L promoter, which are clinically relevant biomarkers for systemic lupus erythematosus (SLE). Through the analysis of 26 patient samples and 14 healthy control samples, EPILIGHT detection results demonstrated complete consistency with pyrosequencing technology. EPILIGHT demonstrated outstanding diagnostic performance, with the sensitivity and specificity of site 1 being 96% and 100%, respectively. The sensitivity and specificity of site 2 were 84.6% and 100%, respectively, which were completely consistent with pyrophosphate sequencing. Meanwhile, the sensitivity and specificity of the EPILIGHT method surpass those of the existing high-resolution melting curve. These results highlight the method’s strong potential for rapid and reliable epigenetic diagnostics with single-CpG resolution.</p>

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EPILIGHT: precise quantification of single-CpG methylation level with a ratiometric CRISPR strategy

  • 期刊论文

  • 发表日期：

    2026年3月9日

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  • Songkuan Zhuang,
  • Ganfeng Deng,
  • Yuzhong Xu,
  • Jiajun Han,
  • Yizhen Liu

摘要

DNA methylation at specific CpG sites serves as a critical biomarker for disease diagnosis, yet existing detection methods often lack single-site resolution, are susceptible to interference from adjacent CpGs, or involve complex and time-consuming processes. Here we introduce EPILIGHT, a rapid and ratiometric CRISPR-based strategy designed for precise quantification of methylation level at individual CpG sites while effectively minimizing interference from flanking CpG sites. The approach integrates ultrafast bisulfite conversion, recombinase polymerase amplification (RPA), and a dual-channel CRISPR/Cas12a detection system to independently recognize methylated (C) and unmethylated (T) sequences. By computing the ratio of methylation-derived signal to the total fluorescence signal via a standard curve, EPILIGHT ensures accurate and reproducible quantification independent of DNA input or reaction efficiency. The entire procedure is completed within 70 min. We validated EPILIGHT by targeting two closely spaced CpG sites in the IFI44L promoter, which are clinically relevant biomarkers for systemic lupus erythematosus (SLE). Through the analysis of 26 patient samples and 14 healthy control samples, EPILIGHT detection results demonstrated complete consistency with pyrosequencing technology. EPILIGHT demonstrated outstanding diagnostic performance, with the sensitivity and specificity of site 1 being 96% and 100%, respectively. The sensitivity and specificity of site 2 were 84.6% and 100%, respectively, which were completely consistent with pyrophosphate sequencing. Meanwhile, the sensitivity and specificity of the EPILIGHT method surpass those of the existing high-resolution melting curve. These results highlight the method’s strong potential for rapid and reliable epigenetic diagnostics with single-CpG resolution.