CRISPR/Cas13a-induced self-priming cyclic amplification enables liquid biopsy of exosomal circular RNA in non-small cell lung cancer

Abstract

The precise and reliable identification of circular RNA (circRNA) is essential for both biological studies and clinical diagnostics of non-small cell lung cancer (NSCLC), especially the exosomal circRNA. In this study, we utilize a CRISPR/Cas13a system to specifically recognize the unique back-splice junction of target circRNA and develop a novel detection platform termed CRISPR/Cas13a-induced self-priming cyclic amplification. This method enables highly sensitive and specific circRNA detection. A pair of stem-loop DNA primers was carefully designed, each incorporating complementary single-stranded DNA sequences and five ribouridine (rU) residues at the 3′ end serving as an overhang. When Cas13a binds to the target circRNA, its trans-cleavage activity is activated, leading to the cleavage of the rU residues. This cleavage permits the 3′ ends of the stem-loop primers to extend along one another, generating multiple double stem-loop DNA structures that initiate successive cycles of self-priming chain elongation. By leveraging the sustained trans-cleavage activity of Cas13a and the high amplification efficiency of the self-priming cyclic reaction, the assay achieves sensitive detection of circRNA at concentrations as low as 564 aM within 90 min. In addition, the proposed method has been successfully applied for the analysis of exosomal hsa_circ_0003026 expression level in normal samples and NSCLC samples and demonstrated the potential of exosomal hsa_circ_0003026 in regulating the pathological progression. Owing to the high specificity of Cas13a, the proposed method can be directly applied to detect circRNA in complex biological samples without prior isolation of corresponding linear RNAs.