Light-Triggered Signal Enhancement Strategy Integrated with a CRISPR/Cas13a-Based Assay for Ultrasensitive and Specific miRNA Detection.

Abstract

The development of facile, accurate, and affordable assays for microRNAs (miRNAs) in early cancer is greatly desirable but encounters an obstacle due to low cellular abundance in biofuids. In this study, we present a novel approach called a light-triggered exponential amplification strategy coupled with a CRISPR/Cas13a-based diagnostic system (LEXPA-CRISPR), which directly transduces rare miRNA targets into photocontrolled signal enhancement response. This innovative platform leverages trans-cleavage of CRISPR/Cas13a, activated by the miRNA target, to cleave specific RNA fragments within the MB@PC-NAC assembly, thus releasing free PC-single-stranded DNA (PC-ssDNA) that is modified by a photocleavable linker (PC linker). UV irradiation is further employed toward the photoresponsive PC-ssDNA, resulting in instantaneous generation of oligo with a new 5' phosphate group (Pho-ssDNA). The Pho-ssDNA serves as a trigger for rolling circle amplification (RCA) reaction, which generates thousands of long ssDNA repeats of diverse lengths with a strong fluorescence signal. Through optimization, we achieved a detection limit of 1 fM for miR21 without the need for target amplification. Moreover, the programmable versatility of LEXPA-CRISPR is also demonstrated for miR17 determination only with simple modification of CRISPR RNA (crRNA) sequences. This proposed biosensor successfully monitored the levels of miR21 and miR17 in tumor cells, showing a satisfactory consistency with the standard qRT-PCR method. Conclusively, LEXPA-CRISPR represents a promising strategy for ultrasensitive miRNA detection. It combines the advantages of light-triggered signal amplification and robust collateral cleavage activity of Cas13a, making it an attractive tool for practical CRISPR-based diagnostics.