A split crRNA with CRISPR-Cas12a enables highly sensitive, selective, and multiplexed detection of RNA and DNA

Abstract

Abstract The complete 40-nt CRISPR RNA (crRNA) of Cas12a can be artificially divided into two parts, including the 20-nt scaffold RNA with fixed sequences and the 20-nt spacer RNA with variable sequences, respectively. Herein, we found that Cas12a protein, scaffold RNA, and spacer RNA can be reassembled into an active ribonucleoprotein (RNP) which has a trans-cleavage activity comparable to that of wild-type Cas12a RNP. By leveraging such split CRISPR-Cas12a system (SCas12a), we devised fast fluorescence and lateral flow assays for highly sensitive, selective, and multiplexed detection of miRNAs without the need for reverse transcription and pre-amplification, achieving a limit of detection (LoD) of 10 fM. Additionally, our SCas12a assay enables detecting long-stranded RNA without secondary structure, as well as distinguishing mature miRNA from its precursor (pre-miRNA) that comprises the same sequence of miRNA. Beyond RNA detection, SCas12a outperforms wild-type Cas12a in specificity towards DNA point mutations. In combination with recombinase polymerase amplification (RPA), we set up a one-pot assay to detect attomolar concentrations of human papillomavirus (HPV) in patient samples. In conclusion, this work provides a simple, cost-effective, yet powerful SCas12a-based rapid nucleic acid detection platform in various diagnostic settings.