Foldback-crRNA-Enhanced CRISPR/Cas13a System (FCECas13a) Enables Direct Detection of Ultrashort sncRNA

Abstract

The CRISPR/Cas13a system has promising applications in clinical small noncoding RNA (sncRNA) detection because it is free from the interference of genomic DNA. However, detecting ultrashort sncRNAs (less than 20 nucleotides) has been challenging because the Cas13a nuclease requires longer crRNA− target RNA hybrids to be activated. Here, we report the development of a foldback-crRNA-enhanced CRISPR/Cas13a (FCECas13a) system that overcomes the limitations of the current CRISPR/Cas13a system in detecting ultrashort sncRNAs. The FCECas13a system employs a 3′-terminal foldback crRNA that hybridizes with the target ultrashort sncRNA, forming a double strand that "tricks" the Cas13a nuclease into activating the HEPN structural domain and generating trans-cleavage activity. The FCECas13a system can accurately detect miRNA720 (a sncRNA currently known as tRNA-derived small RNA), which is only 17 nucleotides long and has a concentration as low as 15 fM within 20 min. This FCECas13a system opens new avenues for ultrashort sncRNA detection with significant implications for basic biological research, disease prognosis, and molecular diagnosis.

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

“…Engineering crRNA modi cations has aimed at lowering detection thresholds 39,41 and improving SNP detection sensitivity 40,42 , focusing particularly on altering the DR sequence's stem-loop structure. Nguyen et al demonstrated the necessity of the stem region for Cas12a's functionality, in contrast to the dispensable loop region.…”