RT-RPA-assisted scaffold RNA transcription amplification activation Cas12a trans-cleavage strategy for one-pot miRNA detection

Abstract

Split crRNA is utilized in the CRISPR system as a highly specific and sensitive tool for nucleic acid identification in molecular diagnostics. Here, we introduce a novel one-pot RT-RPA-assisted Scaffold RNA Transcription Amplification Activation Cas12a Trans-cleavage (ScRNA-TAAT) strategy for miRNA detection. Capable of completing miRNA detection in 50 min with a detection limit of 2.66 aM. The miRNA was amplified into double-stranded DNA amplicons with a T7 promoter using RT-RPA, which is subsequently transcribed into RNA trigger. A split T7 promoter extension sequence with template binds to the RNA trigger, assembly a three-way linker to initiate transcription of the scaffold RNA. The three-way linker with sticky ends joins to its RNA products, assembling a DNA-RNA complex that acts as both a spacer RNA and an activator. Simultaneously, the DNA-RNA hybrid complexes and scaffold RNA could replace the split crRNA and combined with Cas12a, forming an active ribonucleoprotein (RNP) complex with trans-cleavage activity comparable to that of the wild-type Cas12a RNP. Furthermore, we have successfully integrated the ScRNA-TAAT strategy into lateral flow analysis, enabling the visual detection of miRNAs. The assay's exceptional specificity and universality are highlighted by its application to complex biological matrices, including serum and cell lysates. The simplicity, sensitivity, and adaptability of the ScRNA TAAT assay render it a promising candidate for point-of-care testing and high sensitivity in molecular diagnostics.