Target-activated CRISPR/Cas12a recognize multifunctional G-quadruplex and dual fluorescent indicators enable rapid non-extraction analysis of circulating tumor DNA in breast cancer

Abstract

A simple, efficient, and cost-effective method for identifying extremely low levels of circulating tumor DNA (ctDNA) is crucial for the diagnosis and monitoring of cancer. Herein, we designed a label-free and one-pot assay for accurate detection of ctDNA with the combination of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system, G-quadruplex (G4), and dual-signal reporters, polydopamine (PDA) and N-Methyl Mesoporphyrin IX (NMM). Without ctDNA extraction, CRISPR/Cas12a could directly recognize the target double-stranded ctDNA and activate the Cas12a protein. G4 could bind to NMM/K⁺ to enhance its fluorescence signal, and combine with hemin/K⁺ to form a complex that catalyzed dopamine to form PDA and exhibit an intense signal. When the target ctDNA was introduced, the trans-cleavage of G4 by activated Cas12a caused a significant signal decrease in NMM and PDA, acting as a signal amplifier. This assay could detect ctDNA as low as 4 aM in 40 min and distinguish single-base mutations. The CRISPR/Cas12a-G4 biosensor, validated on 48 samples, achieved a specificity of 100 %, a sensitivity of 92 %, and an area under the receiver operating characteristic curve (AUC) of 0.983. These findings were consistently supported by clinical data (imaging results, pathological assessments), and quantitative real-time polymerase chain reaction (qRT-PCR) data. The biosensor's robust performance and potential for application in low-resource settings make it a promising tool for advancing cancer diagnostics and management.