CRISPR-Cas12a/Cas13a Multiplex Bioassay for ctDNA and miRNA by Mass Spectrometry

The CRISPR-Cas system, particularly CRISPR-Cas12a and CRISPR-Cas13a, has been widely utilized in constructing various biosensors due to their "<i>trans</i>-cleavage" ability as a means of signal amplification. However, this universal "<i>trans</i>-cleavage" characteristic also presents a challenge for realizing CRISPR-Cas multiplexed bioanalysis. Besides, potential signal cascading interference and complicated design are notable obstacles in CRISPR-Cas multiplexed bioanalysis. Herein, we propose a mass spectrometry method that leverages the CRISPR-Cas12a/13a system to achieve simultaneous detection of ctDNA and miRNA. Based on the properties of the CRISPR-Cas12a/13a system, two types of nanoparticle reporter probes have been engineered, using cancer-related biomarkers ctDNA and miR-21 as our model analytes. The nanoparticle tags, which intrinsically incorporated millions of detectable atoms, combined with the CRISPR-Cas12a/Cas13a system's "<i>trans</i>-cleavage" ability, allow the proposed mass spectrometry strategy to achieve fmol-level detection limits without any nucleic acid amplification procedures. The assay was successfully applied to human serum samples, demonstrating its potential for early disease diagnosis and progression tracking.