Single-molecule assay guided crRNA optimization enhances specific microRNA detection by CRISPR-Cas12a

CRISPR-Cas12a is a promising tool for nucleic acid detection. However, due to the protein flexibility, Cas12a tolerates mismatches, which limits its specificity. In this study, the single-molecule assay revealed that the length of crRNA regulates the association kinetics between crRNA/Cas12a complex and target DNA. Short-crRNA/ Cas12 associates the target dsDNA 2-fold faster than the single-nucleotide mismatched dsDNA, whereas the long-crRNA/Cas12a binds to the fully matched and single-nucleotide mismatched DNA targets with similar rates. These findings are further corroborated by electrophoretic mobility shift assay (EMSA) and double-stranded DNA (dsDNA) cleavage results. Inspired by these findings, we established stem-loop amplification conjugated short crRNA CRISPR-Cas12a (SlashCas12a) detection. In this approach, stem-loop-mediated microRNA reverse transcription was harnessed to enhance the amplification efficiency of the short single-stranded RNA. The shortcrRNA Cas12a specifically distinguishes the single mismatched miRNA homologs, i.e. let-7a family members. The introduction of PAM containing stem-loop can break through the limitation of PAM for Cas12a and efficiently amplify the miRNAs. Multiple miRNAs, including miR-122b, miR-21, and let-7a, can be efficiently detected, and the limit of detection is up to 7.8 aM. Furthermore, the distinct expression of miR-21 and let-7a can be detected in the lung adenocarcinoma and breast samples using this method. These results demonstrate the appropriate crRNA engineering will extend the application of Cas12a in the specific molecule diagnosis.