Label-Free and Universal CRISPR/Cas12a-Based Detection\nPlatform for Nucleic Acid Biomarkers

CRISPR/Cas12a\nhas been widely used in molecular diagnostics due\nto its excellent trans-cleavage activity. However, conventional reporters,\nsuch as F/Q-labeled single-stranded DNA (ssDNA) reporters, enzyme-labeled\nreporters, and spherical nucleic acid reporters, require complex modification\nor labeling processes. In this study, we have developed a rapid, universal,\nand label-free CRISPR/Cas12a-based biomarker detection platform via\ndesigning a G-quadruplex (G4) containing a hairpin structure as the\nreporter. The hairpin loop design of hairpin G4 improves the cleavage\nefficiency of Cas12a and the signal strength of the G4 binding ligand.\nMeanwhile, the incorporation of a G4 binding dye (protoporphyrin IX)\neliminates the need for complex modifications. The CRISPR-hairpin\nG4 detection platform is capable of detecting ssDNA, double-stranded\nDNA, genetic RNAs, and miRNAs. Moreover, this platform achieves label-free\ndetection in clinical samples, demonstrating its practical applicability\nand efficiency.