School of Chemistry and Biological Engineering, University of Science and Technology Beijing, 30 Xueyuan Road, Haidian District, Beijing, 100083, China.
School of Chemistry and Biological Engineering, University of Science and Technology Beijing, 30 Xueyuan Road, Haidian District, Beijing, 100083, China.
Talanta. 2022 Aug 15;246:123521. doi: 10.1016/j.talanta.2022.123521. Epub 2022 May 3.
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.
快速、灵敏和特异性地测定环状 RNA（circRNA）对于生物研究和临床诊断都具有重要意义。由于大量相应的线性 RNA 与 circRNA 的共同存在，并且除了 circRNA 的连接序列外，它们具有相同的序列，因此对靶 circRNA 的特异性识别目前面临重大挑战。在此，我们首次利用 CRISPR/Cas13a 特异性识别靶 circRNA 的独特连接序列，并创新性地开发了一种用于灵敏和特异性检测 circRNA 的 CRISPR/Cas13a 诱导指数扩增检测法。一对茎环 DNA 引物经过精心设计，在其 3' 末端具有一对互补的单链 DNA 和五个尿嘧啶核糖核苷酸作为悬臂。一旦 Cas13a 识别靶 circRNA，Cas13a 的转切割活性就会被激活，尿嘧啶核糖核苷酸将被切割。因此，茎环引物的 3' 末端可以彼此延伸，产生大量可以引发多个环介导等温扩增（LAMP）的双链茎环 DNA。利用 Cas13a 的持续切割活性和多个 LAMP 反应的高扩增效率，可以在 30 分钟内灵敏地检测低至 1 fM 的靶 circRNA。由于 Cas13a 的高度特异性，该测定法已成功应用于真实生物样本中 circRNA 的检测，而无需分离相应的线性 RNA。此外，该测定法为所有序列特异性 RNA 靶标的检测提供了一个通用平台，表明我们的 CRISPR/Cas13a 诱导指数扩增检测法在基础研究和临床诊断中用于 RNA 生物标志物的检测具有很大的潜力。
