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Allele-Specific CRISPR-Cas9-Based Ratiometric Fluorescence Platform for Portable EGFR L858R Mutation Detection.

Wenjun Ming1, Yidan Zhu1, Longjie Li1

  • 1Jiangsu Key Laboratory of Advanced Medical Analysis and Public Health, Nantong Key Laboratory of Public Health and Medical Analysis, School of Public Health, Nantong University, Nantong, Jiangsu 226019, P. R. China.

Analytical Chemistry
|November 12, 2025
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Summary

A new CRISPR-Cas9 sensor detects low-abundance EGFR L858R mutations in circulating tumor DNA (ctDNA) for early non-small cell lung cancer (NSCLC) diagnosis. This portable, visual point-of-care test achieves high accuracy, aiding resource-limited settings.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Medical Diagnostics

Background:

  • Early detection of non-small cell lung cancer (NSCLC) relies on identifying specific mutations like EGFR L858R in circulating tumor DNA (ctDNA).
  • Low abundance of ctDNA poses a challenge for sensitive and specific mutation detection.
  • Current diagnostic methods may lack portability and cost-effectiveness for widespread screening.

Purpose of the Study:

  • To develop a portable CRISPR-Cas9-based ratiometric fluorescence sensor for sensitive detection of the EGFR L858R mutation.
  • To enable visual point-of-care testing (POCT) for early NSCLC diagnosis.
  • To provide a cost-effective and accessible screening solution, especially for resource-limited settings.

Main Methods:

  • Engineered sgRNA to position the L858R mutation within the Cas9 PAM region for allele-specific activation.
  • Utilized trans-cleavage of blocker DNAs leading to increased Cy5 fluorescence.
  • Incorporated a Pepper aptamer and RNA interaction to cause decreased HBC-530 fluorescence, creating a dual-channel ratiometric readout.
  • Developed a compact 3D-printed device for visual POCT.

Main Results:

  • Achieved visual POCT of L858R mutation with single-nucleotide specificity at an allele frequency as low as 0.01%.
  • Demonstrated 100% concordance with clinical diagnoses in 22 plasma samples.
  • The ratiometric fluorescence sensor provided a dual-channel readout for enhanced accuracy.

Conclusions:

  • The portable CRISPR-Cas9 sensor offers an effective, cost-effective, and accessible method for early NSCLC screening.
  • The developed platform enables sensitive detection of low-abundance EGFR L858R mutations at the point of care.
  • This technology holds significant potential for improving NSCLC diagnosis in diverse healthcare environments.