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Rolling Circular Amplification (RCA)-Assisted CRISPR/Cas9 Cleavage (RACE) for Highly Specific Detection of Multiple Extracellular Vesicle MicroRNAs.
Analytical Chemistry ( IF 7.4 ) Pub Date : 2020-01-09 , DOI: 10.1021/acs.analchem.9b04814
Ruixuan Wang 1, 2, 3 , Xianxian Zhao 3 , Xiaohui Chen 2, 4 , Xiaopei Qiu 2, 3 , Guangchao Qing 2, 4 , Hong Zhang 2, 4 , Liangliang Zhang 2, 4 , Xiaolin Hu 2, 4 , Zhuoqi He 4 , Daidi Zhong 4 , Ying Wang 5 , Yang Luo 2, 4
Affiliation  

Multiplexed detection of extracellular vesicle (EV)-derived microRNAs (miRNAs) plays a critical role in facilitating disease diagnosis and prognosis evaluation. Herein, we developed a highly specific nucleic acid detection platform for simultaneous quantification of several EV-derived miRNAs in constant temperature by integrating the advantages of a clustered regularly interspaced short palindromic repeats/CRISPR associated nucleases (CRISPR/Cas) system and rolling circular amplification (RCA) techniques. Particularly, the proposed approach demonstrated single-base resolution attributed to the dual-specific recognition from both padlock probe-mediated ligation and protospacer adjacent motif (PAM)-triggered cleavage. The high consistency between the proposed approach RCA-assisted CRISPR/Cas9 cleavage (RACE) and reverse transcription quantitative polymerase chain reaction (RT-qPCR) in detecting EV-derived miRNAs' abundance from both cultured cancer cells and clinical lung cancer patients validated its robustness, revealing its potentials in the screening, diagnosis, and prognosis of various diseases. In summary, RACE is a powerful tool for multiplexed, specific detection of nucleic acids in point-of-care diagnostics and field-deployable analysis.

中文翻译:

滚动循环扩增(RCA)辅助的CRISPR / Cas9裂解(RACE),用于多种细胞外囊泡MicroRNA的高特异性检测。

细胞外小泡(EV)衍生的microRNA(miRNA)的多重检测在促进疾病诊断和预后评估中起着关键作用。在此,我们开发了一种高度特异性的核酸检测平台,通过整合成簇的规则间隔的短回文重复序列/ CRISPR相关核酸酶(CRISPR / Cas)系统和滚动循环扩增( RCA)技术。特别地,所提出的方法证明了单碱基分辨力归因于来自挂锁探针介导的连接和原间隔子相邻基序(PAM)触发的裂解的双重特异性识别。拟议的方法RCA辅助的CRISPR / Cas9裂解(RACE)和逆转录定量聚合酶链反应(RT-qPCR)在从培养的癌细胞和临床肺癌患者中检测EV衍生的miRNA丰度方面具有高度一致性,揭示了其在各种疾病的筛查,诊断和预后方面的潜力。总而言之,RACE是在即时诊断和现场可部署的分析中对核酸进行多重,特异性检测的强大工具。
更新日期:2020-01-09
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