Harnessing noncanonical trans-cleavage characteristics of Cas12 and Cas13a to enhance CRISPR-based diagnostics

Cas12 and Cas13 are extensively utilized in molecular diagnostics for their trans -cleavage activities, yet their activation characteristics remain partially understood. Here, we conduct an in-depth investigation of Cas12a, Cas12f1, and Cas13a, uncovering the characteristics of their trans -DNase and trans -RNase activities with noncanonical activators. Our findings reveal that DNA can serve as a direct target for CRISPR-Cas13a, markedly increasing the detection sensitivity for single-base mismatches. Moreover, the trans -cleavage activities of Cas12a and Cas13a can be activated by diverse RNA:DNA and RNA:RNA duplexes, respectively, indicating that the presence of stem–loop structures in crRNAs is not essential for their activation. Notably, Cas12f1, unlike Cas12a, exhibits intrinsic RNase activity independently of activation. Leveraging these insights, we have improved the accuracy of a dual-gene target detection approach that employs the CRISPR-Cas12f1 and Cas13a systems. Our research advances the understanding of the noncanonical activation characteristics of Cas12 and Cas13a, contributing to the field of CRISPR-based diagnostics. Uncovering noncanonical trans -cleavage characteristics of Cas12 and Cas13a improves the accuracy of CRISPR-based dual-gene diagnostics and enhances detection sensitivity for single base mismatches through newly discovered activation mechanisms