文献类型：期刊文献

英文题名：An RNA-based catalytic hairpin assembly circuit coupled with CRISPR-Cas12a for one-step detection of microRNAs

作者：Chen, Pinru[1];Wang, Luying[1];Qin, Peipei[1];Yin, Bin-Cheng[1,2,3];Ye, Bang-Ce[1,2]

机构：[1]Zhejiang Univ Technol, Coll Pharmaceut Sci, Collaborat Innovat Ctr Yangtze River Delta Reg Gr, Inst Engn Biol & Hlth, Hangzhou 310014, Zhejiang, Peoples R China;[2]East China Univ Sci & Technol, Lab Biosyst & Microanal, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[3]Shihezi Univ, Sch Chem & Chem Engn, Shihezi 832000, Xinjiang, Peoples R China

年份：2022

卷号：207

外文期刊名：BIOSENSORS & BIOELECTRONICS

收录：;EI(收录号：20221211816916);WOS:【SCI-EXPANDED(收录号:WOS:000789678700003)】；

基金：This work was jointly supported by the National Natural Science Foundation of China (Grants 21822402, 22134003), the National Key R&D Program of China (Grant 2020YFA0908800), "Shuguang Program" supported by Shanghai Education Development Foundation and Shanghai Municipal Education Commission, and the Joint Funds of the Zhejiang Provincial Natural Science Foundation of China (Grant LHDMZ22H300008).

语种：英文

外文关键词：MicroRNA detection; Catalytic hairpin assembly; RNA circuit; Cas12a; Nuclease activity

摘要：CRISPR-Cas nuclease-based nucleic acid detection has exhibited extraordinary value in the field of molecular diagnostics, but it usually involves two separate reaction steps of nucleic acid amplification and Cas-based endpoint detection, resulting in the use of multiple enzymes, inconvenient operation, and potential carry-over contamination. Here, we propose an RNA-based catalytic hairpin assembly (CHA) circuit coupled with CRISPR-Cas12a for one-step detection of microRNAs (miRNAs) at an isothermal condition. This method relies on the rational design of a spacer-blocking crRNA as a bridge between the two systems. The target miRNA can specifically trigger RNA-based CHA and induce a configurational change of the blocked crRNAs into precursor crRNAs (pre-crRNAs), which can be processed into mature crRNAs to function by leveraging the inherent RNase activities of Cas12a. In this way, the developed circuit achieves a femtomolar detection limit and shows an accurate detection of miRNA levels in different cell lines. Therefore, our method would provide a new paradigm to develop miRNA detection methods based on the CRISPR/Cas system.