New CRISPR-Derived microRNA Sensing Mechanism Based on Cas12a Self-Powered and Rolling Circle Transcription-Unleashed Real-Time crRNA Recruiting

Abstract

Current CRISPR-Cas-based nucleic acid sensing methods relying on the preassembled Cas-crRNA complexes are generally limited to the detection of protospacer-adjacent motif (PAM)-containing sequences, and nonspecific backgrounds are inevitable. Herein, we propose a new CRISPR-derived microRNA sensing mechanism based on rolling circle transcription (RCT)-unleashed self-recruiting of crRNA by Cas12a (Cas12a-SCR). In Cas12a-SCR, target microRNA can specifically trigger RCT to produce a long single- strand RNA with numerous pre-crRNA repeats, which can be trimmed and recruited by Cas12a actively. This new target-initiated, real-time producing, trimming, and self-assembling manner of Cas12a-crRNA remarkably suppresses the nonspecific background and relieves the stringent requirement of PAM site in the target sequence. Thus, the universality of the Cas12a-SCR toward different nucleic acid sequences is greatly expanded.