CRISPR/Cas13a-triggered Cas12a biosensing method for ultrasensitive and specific miRNA detection

作者全名："Zhao, Dan; Tang, Jiutang; Tan, Qin; Xie, Xiaohong; Zhao, Xin; Xing, Dingpei"

作者地址："[Zhao, Dan; Xie, Xiaohong; Zhao, Xin] Chongqing Univ Three Gorges Hosp, Dept Clin Lab, Chongqing 404000, Peoples R China; [Tang, Jiutang] Chongqing Med Univ, Ctr Neurosci Res, Chongqing 400010, Peoples R China; [Tan, Qin] Chongqing Wanzhou Shanghai Hosp, Dept Clin Lab, Chongqing 404000, Peoples R China; [Xing, Dingpei] Chongqing Univ Three Gorges Hosp, Dept Pediat Surg, Chongqing 404000, Peoples R China"

通信作者："Xing, DP (通讯作者)，Chongqing Univ Three Gorges Hosp, Dept Pediat Surg, Chongqing 404000, Peoples R China."

来源：TALANTA

ESI学科分类：CHEMISTRY

WOS号：WOS:000990538400001

JCR分区：Q1

影响因子：5.6

年份：2023

卷号：260

期号：　

开始页：　

结束页：　

文献类型：Article

关键词：CRISPR-Cas13a; CRISPR-Cas12a; MiRNA-155 detection; Fluorescent sensing strategy

摘要："Constructing an ultrasensitive CRISPR/Cas-based biosensing strategy is highly significant for the detection of trace targets. Here we presented a dual-amplified biosensing method based on CRISPR/Cas13a-triggered Cas12a, namely, Cas13a-12a amplification. As proof-of-principle, the developed strategy was used for miRNA-155 detection. The target bound to the Cas13a-crRNA complex and activated the cleavage activity of Cas13a for cleaving uracil ribonucleotides (rU) in the bulge structure of blocker strand (BS), resulting in the release of primer strand (PS) from the BS modified on magnetic beads. Then, the released PS activated the cleavage activity of Cas12a to cleave single-strand DNA reporter probes, producing a significantly increased fluorescent signal. The detection limit of the Cas13a-12a amplification using synthetic miRNA-155 was as low as 0.35 fM, which was much lower than that of the only Cas13a-based assay. The applied performance of this amplification strategy was verified by accurately quantifying miRNA-155 expression levels in different cancer patients. Therefore, the developed strategy offers a supersensitive and highly specific miRNAs sensing platform for clinical application."

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