Unraveling the influence of CRISPR/Cas13a reaction components on enhancing trans-cleavage activity for ultrasensitive on-chip RNA detection

被引:2
|
作者
He, Qian [1 ,2 ]
Chen, Qun [2 ]
Lian, Lijin [2 ]
Qu, Jiuxin [3 ]
Yuan, Xi [2 ]
Wang, Chuhui [2 ]
Xu, Lidan [2 ,4 ]
Wei, Jiazhang [5 ]
Zeng, Shaoling [6 ]
Yu, Dongmei [7 ]
Dong, Yuhan [2 ]
Zhang, Yongbing [8 ]
Deng, Lin [9 ]
Du, Ke [10 ]
Zhang, Canyang [2 ]
Pandey, Vijay [2 ]
Gul, Ijaz [1 ,2 ]
Qin, Peiwu [1 ,2 ]
机构
[1] Hangzhou Dianzi Univ, Sch Commun Engn, Hangzhou 310018, Zhejiang, Peoples R China
[2] Tsinghua Univ, Tsinghua Shenzhen Int Grad Sch, Inst Biopharmaceut & Hlth Engn, Shenzhen 518055, Guangdong, Peoples R China
[3] Shenzhen Third Peoples Hosp, Clin Lab, Shenzhen 518115, Guangdong, Peoples R China
[4] Harbin Med Univ, Lab Med Genet, Harbin 150081, Peoples R China
[5] Guangxi Acad Med Sci, Peoples Hosp Guangxi Zhuang Autonomous Reg, Dept Otolaryngol & Head & Neck, 6 Taoyuan Rd, Nanning 530021, Peoples R China
[6] Shenzhen Customs, Anim & Plant Inspect & Quarantine Technol Ctr, Shenzhen, Peoples R China
[7] Shandong Univ, Sch Mech Elect & Informat Engn, Weihai 264209, Shandong, Peoples R China
[8] Harbin Inst Technol Shenzhen, Shenzhen 518055, Peoples R China
[9] Shenzhen Bay Lab, Inst Mol Physiol, Shenzhen 518132, Peoples R China
[10] Univ Calif Riverside, Chem & Environm Engn, Riverside, CA USA
基金
中国国家自然科学基金;
关键词
CRISPR/Cas13a; TIRFM; RNA detection; Amplification-free; crRNA combination; NUCLEIC-ACID DETECTION; SARS-COV-2;
D O I
10.1007/s00604-024-06545-4
中图分类号
O65 [分析化学];
学科分类号
070302 ; 081704 ;
摘要
The CRISPR/Cas13 nucleases have been widely documented for nucleic acid detection. Understanding the intricacies of CRISPR/Cas13's reaction components is pivotal for harnessing its full potential for biosensing applications. Herein, we report on the influence of CRISPR/Cas13a reaction components on its trans-cleavage activity and the development of an on-chip total internal reflection fluorescence microscopy (TIRFM)-powered RNA sensing system. We used SARS-CoV-2 synthetic RNA and pseudovirus as a model system. Our results show that optimizing Mg2+ concentration, reporter length, and crRNA combination significantly improves the detection sensitivity. Under optimized conditions, we detected 100 fM unamplified SARS-CoV-2 synthetic RNA using a microtiter plate reader. To further improve sensitivity and provide a new amplification-free RNA sensing toolbox, we developed a TIRFM-based amplification-free RNA sensing system. We were able to detect RNA down to 100 aM. Furthermore, the TIRM-based detection system developed in this study is 1000-fold more sensitive than the off-coverslip assay. The possible clinical applicability of the system was demonstrated by detecting SARS-CoV-2 pseudovirus RNA. Our proposed sensing system has the potential to detect any target RNA with slight modifications to the existing setup, providing a universal RNA detection platform.
引用
收藏
页数:12
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