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

Effective detection of low-abundance EGFR L858R mutation from circulating tumor DNA (ctDNA) is critical for early stage NSCLC diagnosis. Here, a portable CRISPR-Cas9-based ratiometric fluorescence sensor was proposed. Typically, allele-specific activation of Cas9 and the trans-cleavage of Cy5/BHQ2-labeled blocker DNAs were achieved by engineering sgRNA to position the L858R mutation within the PAM region of Cas9, resulting in increased Cy5 fluorescence. Simultaneously, the attenuated fluorescence of HBC-530 was observed because the released input RNA bound to the Pepper aptamer, which resulted from the decreased melting temperature ( T m ) of the blocker DNA-RNA hybrid. Thus, a dual-channel ratiometric readout can be readily attained. Ultimately, visual point-of-care testing (POCT) of L858R at 0.01% allele frequency with single-nucleotide specificity was realized using a compact 3D-printed device. The validation result of 22 collected plasma samples demonstrated 100% concordance with the clinical diagnoses. This platform provides a cost-effective and accessible solution for NSCLC screening, making it particularly suitable for resource-limited healthcare settings.