Chen Yong, Zhang Yibin, Luo Siyuan, Yang Xinyao, Liu Conghui, Zhang Qianling, Liu Yizhen, Zhang Xueji
Research Center for Nanosensor Molecular Diagnostic & Treatment Technology, College of Chemistry and Environmental Engineering, Shenzhen University, Shenzhen 518060, Guangdong, P. R. China.
Graphene Composite Research Center, College of Chemistry and Environmental Engineering, Shenzhen University, Shenzhen 518060, Guangdong, P. R. China.
Anal Chem. 2023 Oct 24;95(42):15606-15613. doi: 10.1021/acs.analchem.3c02687. Epub 2023 Oct 12.
The CRISPR/Cas13a system has promising applications in clinical small noncoding RNA (sncRNA) detection because it is free from the interference of genomic DNA. However, detecting ultrashort sncRNAs (less than 20 nucleotides) has been challenging because the Cas13a nuclease requires longer crRNA-target RNA hybrids to be activated. Here, we report the development of a foldback-crRNA-enhanced CRISPR/Cas13a (FCECas13a) system that overcomes the limitations of the current CRISPR/Cas13a system in detecting ultrashort sncRNAs. The FCECas13a system employs a 3'-terminal foldback crRNA that hybridizes with the target ultrashort sncRNA, forming a double strand that "tricks" the Cas13a nuclease into activating the HEPN structural domain and generating -cleavage activity. The FCECas13a system can accurately detect miRNA720 (a sncRNA currently known as tRNA-derived small RNA), which is only 17 nucleotides long and has a concentration as low as 15 fM within 20 min. This FCECas13a system opens new avenues for ultrashort sncRNA detection with significant implications for basic biological research, disease prognosis, and molecular diagnosis.
CRISPR/Cas13a系统在临床小非编码RNA（sncRNA）检测中具有广阔的应用前景，因为它不受基因组DNA的干扰。然而，检测超短sncRNA（少于20个核苷酸）一直具有挑战性，因为Cas13a核酸酶需要更长的crRNA-靶标RNA杂交体才能被激活。在此，我们报告了一种回折式crRNA增强的CRISPR/Cas13a（FCECas13a）系统的开发，该系统克服了当前CRISPR/Cas13a系统在检测超短sncRNA方面的局限性。FCECas13a系统采用3'末端回折式crRNA，它与靶标超短sncRNA杂交，形成一条双链，“欺骗”Cas13a核酸酶激活HEPN结构域并产生切割活性。FCECas13a系统能够在20分钟内准确检测出长度仅为17个核苷酸、浓度低至15 fM的miRNA720（一种目前被称为tRNA衍生小RNA的sncRNA）。这种FCECas13a系统为超短sncRNA检测开辟了新途径，对基础生物学研究、疾病预后和分子诊断具有重要意义。
