CRISPR/Cas13a <i>trans-</i>Cleavage-Driven Programmable Autonomous Synthesis of a Functional G-Quadruplex for Ultrasensitive Detection of Circular RNAs with an Improved Signal-to-Background Ratio

Circular RNAs (circRNAs) are a prevalent class of endogenous RNAs that regulate gene expression in various biological pathways, and their dysregulation is closely linked to tumorigenesis. Herein, we demonstrate the CRISPR/Cas13a <i>trans-</i>cleavage-driven programmable autonomous synthesis of a functional G-quadruplex for ultrasensitive detection of circRNAs in living cells and clinical tissues. Upon specific binding to target circRNA, the activated Cas13a/crRNA enables collateral cleavage of a uracil ribonucleotide (rU)-containing substrate probe, releasing a trigger sequence with a 2',3'-cyclic phosphate at its 3'-end. Subsequently, the resultant trigger sequence initiates primer exchange reaction (PER) cascades after its 3'-end is repaired by T4 polynucleotide kinase (T4 PNK), generating numerous long G-quadruplex sequences with different lengths. The long G-quadruplex sequences can be lighted up by thioflavin T (ThT) to obtain a dramatically amplified fluorescence signal. Owing to the exceptional specificity of high-fidelity CRISPR/Cas13a, high amplification efficiency of PER cascades, and high signal-to-background ratio of the G-quadruplex/ThT complex, this method can achieve ultrasensitive detection of circRNA down to 0.83 aM and discriminate target circRNA from its mismatched variants with single-base resolution. Moreover, it can profile endogenous circRNA in single cancer cell and differentiate circRNA expression in breast cancer tissues and their healthy counterparts. Notably, this assay can be accomplished in a one-step and single-tube manner at physiological temperature with the characteristics of easy design, simplified operation, rapid turnaround, and cost-effectiveness, suitable for in-field molecular diagnostics with limited resources.