ID-CRISPR: A CRISPR/Cas12a platform for label-free and sensitive detection of rare mutant alleles using self-interference DNA hydrogel reporter.

Accurate and sensitive detection of single nucleotide variants (SNVs) is paramount for cancer diagnosis and treatment. The CRISPR/Cas12a system shows promise for SNV detection due to its high sensitivity and single-base specificity. However, most CRISPR/Cas12a-based methods rely on F/Q-labeled single-stranded DNA (ssDNA) reporters, which are susceptible to fluorescence fluctuations, thereby reducing accuracy. To address these limitations, researchers have proposed using DNA hydrogels as signal transducers in CRISPR/Cas12a systems. Yet, the encapsulation of indicators into DNA hydrogels introduces additional instability, which could compromise both detection sensitivity and linearity. In this study, we integrated hyperspectral interferometry into a DNA hydrogel-based CRISPR/Cas12a detection platform (ID-CRISPR) to achieve sensitive label-free SNV detection. Using EGFR L858R SNV as a model target, we demonstrated that ID-CRISPR can detect mutant allele frequencies (MAFs) as low as 0.1% with a limit of detection (LOD) of 5 aM, while also showing its potential for quantifying SNV abundance. Its clinical utility was confirmed through analysis of lung tumor samples, with results consistent with sequencing data. Therefore, ID-CRISPR provides a sensitive, label-free, and user-friendly platform for SNV detection, offering new insights into combining optical sensing with DNA hydrogel technology in CRISPR/Cas assays.