CRISPR/Cas13a induced exponential amplification for highly sensitive and specific detection of circular RNA

Abstract

Rapid, sensitive and specific determination of circular RNA (circRNA) is of great significance for both biological research and clinical diagnosis. Specific recognition of target circRNA is now facing major challenges due to the fact that large amount of corresponding linear RNA is coexisting and possesses the same sequences except the junction sequence of circRNA. Herein, we firstly utilize CRISPR/Cas13a to specifically recognize the unique junction sequence of target circRNA and innovatively develop a CRISPR/Cas13a induced exponential amplification assay for sensitive and specific detection of circRNA. A pair of stem-loop DNA primers are elaborately designed with a pair of complementary single-strand DNA and five uracil ribonucleotides as the cantilever at their 3′ terminus. Once Cas13a recognizes target circRNA, the trans-cleavage activity of Cas13a can be activated and the uracil ribonucleotides will be cleaved. Thus, the 3′ terminus of the stem-loop primers can extend along each other to generate a lot of double stem-loop DNAs which can initiate multiple loop-mediated isothermal amplification (LAMP). Taking advantage of the incessant cleavage activity of Cas13a and the high amplification efficiency of multiple LAMP reaction, as low as 1 fM target circRNA can be sensitively detected within 30 min. Due to the high specificity of Cas13a, the proposed assay has been successfully applied to the detection of circRNA in real biological samples without separation of corresponding linear RNAs. Moreover, the proposed assay has offered a versatile platform for the detection of all sequence-specific RNA targets, indicating that our CRISPR/Cas13a induced exponential amplification assay has great potential for the detection of RNA biomarkers in both fundamental studies and clinical diagnostics.