T7 RNA polymerase-mediated rolling circle transcription and the CRISPR-Cas13a cascade reaction for sensitive and specific detection of piRNA.

The aberrant expression of piRNAs in germ cells is a potential cause of male infertility. Establishing diagnostic methods with highly specific biomarkers for male infertility is important for accurate diagnosis and treatment of male infertility. In this study, we proposed a novel method combining rolling circle transcription (RCT) and Cas13a techniques, which utilized the high amplification efficiency of RCT and the two different RNase activities possessed by Cas13a, establishing a highly sensitive and specific assay for male infertility-associated piRNA. First, a circular DNA template was synthesized by hybridizing linear ssDNA with the T7 promoter. The nick in the circular DNA was closed by T4 DNA ligase. In the presence of T7 RNA polymerase, the closed circular DNA produced tandemly repeated pre-crRNA. The RNase activity of Cas13a was used to process pre-crRNAs to form mature crRNA. Guided by crRNA, Cas13a specifically recognized piRNA and activated collateral activity. Activated Cas13a disaggregated thousands of fluorescent probes for each target RNA detected, resulting in powerful signal amplification. As a proof of concept, piR-hsa-14 was used as the validation target. The limit of detection was as low as 3.32 fM with a good linearity in the range of 100 fM to 50 pM. Recovery of piR-hsa-14 ranged from 91.33% to 112.63% in spiked recovery experiments using human serum samples. The results revealed that this method has the advantages of high sensitivity, sufficient accuracy and good reproducibility. We believe that this method could have a promising future as a potential tool for clinical diagnosis of male infertility.