Reducing the Concentration of Hairpins to Weaken Their Nonspecific Adsorption on Cas12a for a Seven-Order-of-Magnitude Reduction in the Limit of Detection

The trans-cleavage activity of Cas12a is affected by crRNA, Reporter, buffer components, and so on, which dominate the sensitivity of the detection method. Herein, we found that the concentration of DNA in the reaction system also affected the trans-cleavage activity of Cas12a. This work proposed a protocol DExo-Cas12a combining dual Exonuclease III (Exo III) and Cas12a/crRNA to achieve triple signal amplification and detection of Cu/Zn SOD mRNA. The target undergoes dual-Exo III-assisted amplification and transforms into numerous cleavage products P2, which acts as an activator of Cas12a to activate trans-cleavage activity. The limit of detection (LOD) in the presence of 20 nM hairpins obtained through condition optimization is 5.4 pM, which is similar to the sensitivity of Cas12a itself. After reducing the concentration of hairpins by 10 times, the LOD decreased to 0.4 aM because high concentrations of hairpins nonspecifically adsorbed on the Cas12a protein and inhibited its activity. This assay exhibited excellent sensitivity and selectivity through a reasonable integration of three amplification processes. Finally, the proposed assay could detect targets in 0.92 ng of total RNA extracts from MCF-7 cells and showed significant differences compared to MCF-10A. The proposed protocol has the potential to become a diagnostic tool for diseases.