Controlling the trans-cleavage of CRISPR-Cas12a with nicked PAM: Universal platform for biosensing

CRISPR-Cas12a is able to precisely recognize double-stranded DNA (dsDNA) containing PAM sequence under the guidance of CRISPR RNA (crRNA) and release indiscriminate trans-cleavage activity, providing an efficient signal amplification tool for molecular diagnostics. In this study, we investigated the trans-cleavage activity of CRISPR-Cas12a in detail by engineering nicked dsDNA activators with different nucleotide defective modes. The influ-ence of PAM nucleotides on the activation of CRISPR-Cas12a trans-cleavage activity and the process by which Cas12a-enhanced strand displacement mechanism to promote crRNA invasion have been revealed. Meanwhile, we demonstrated that dsDNA with a nicked PAM could be specifically recognized and initiate the trans-cleavage activity of CRISPR-Cas12a efficiently. Based on binding-induced PAM assembly, we developed a modular CRISPR-Cas12a-based signal reporting system for universal biosensing. Since different kinds of targets can be recognized with corresponding affinity probes, this system allows the general detection of a wide range of analytes. As a proof of concept, the system was employed for intracellular BCR/ABL1 mRNA imaging and PDGF-BB detection with high sensitivity and specificity. This work is of great importance for the fundamental un-derstanding of CRISPR-Cas12a system and provides significant improvements for the rational design of CRISPR-Cas12a-based sensing platform for molecular diagnostic applications.