Pre-Folded G-Quadruplex as a Tunable Reporter to Facilitate CRISPR/Cas12a-Based Visual Nucleic Acid Diagnosis
作者全名："Yang, Tiantian; Li, Juan; Zhang, Decai; Cheng, Xiaoxue; Li, Jia; Huang, Xiaolin; Ding, Shijia; Tang, Ben Zhong; Cheng, Wei"
作者地址："[Yang, Tiantian; Zhang, Decai; Cheng, Xiaoxue; Li, Jia; Cheng, Wei] Chongqing Med Univ, Affiliated Hosp 1, Ctr Clin Mol Med Detect, Chongqing 400016, Peoples R China; [Li, Juan; Huang, Xiaolin] Nanchang Univ, Sch Food Sci & Technol, State Key Lab Food Sci & Technol, Nanchang 330047, Peoples R China; [Tang, Ben Zhong] Chinese Univ Hong Kong, Shenzhen Inst Aggregate Sci & Technol, Sch Sci & Engn, Shenzhen 518172, Guangdong, Peoples R China; [Ding, Shijia] Chongqing Med Univ, Coll Lab Med, Key Lab Clin Lab Diagnost, Minist Educ, Chongqing 400016, Peoples R China"
通信作者："Cheng, W (通讯作者)，Chongqing Med Univ, Affiliated Hosp 1, Ctr Clin Mol Med Detect, Chongqing 400016, Peoples R China.; Li, J; Huang, XL (通讯作者)，Nanchang Univ, Sch Food Sci & Technol, State Key Lab Food Sci & Technol, Nanchang 330047, Peoples R China.; Tang, BZ (通讯作者)，Chinese Univ Hong Kong, Shenzhen Inst Aggregate Sci & Technol, Sch Sci & Engn, Shenzhen 518172, Guangdong, Peoples R China."
文献类型：Article; Early Access
关键词：G-quadruplex; CRISPR; Cas12a; label-free; signal-on; visual nucleic acid diagnosis
摘要："Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a-based detection strategies with a fluorophore quencher-labeled ssDNA reporter or gold nanoparticle ssDNA reporter have been widely used in point-of-care (POC) molecular diagnostics. However, the potential of these CRISPR/Cas12a strategies for POC molecular diagnostics is often compromised due to the complex labeling, high cost, and low signal-to-noise ratio. Herein, we show a pre-folded G-quadruplex (G4) structure with tunable tolerance to CRISPR/Cas12a trans-cleavage and explore its mechanism. Two G4 structures (i.e., Tel22-10 and G16C) sensitive or tolerant to CRISPR/Cas12a trans-cleavage are designed and used as signal elements to fabricate a label-free visible fluorescent strategy or ""signal-on"" colorimetric strategy, respectively. These two strategies facilitate an ultrasensitive visual nucleic acid determination of Group B Streptococci with a naked-eye limit of detection of 1 aM. The feasibility of the developed G4-assisted CRISPR/Cas12a strategies for real-world applications is demonstrated in clinical vaginal/anal specimens and further verified by a commercial qPCR assay. This work suggests that the proposed G4 structures with tunable tolerance can act as promising signal reporters in the CRISPR/Cas12a system to enable ultrasensitive visible nucleic acid detection."
基金机构：Natural Science Foundation of China; Foundation for Innovative Research Groups of Chongqing Higher Education Institutions; Chongqing Science Fund for Distinguished Young Scholars; Chongqing Talents-Innova-tion Leading Talents Project; ; ; ; [CXQT20013]; [cstc2019jcyjjqX0028]; [CQYC20200303107]
基金资助正文："ACKNOWLEDGMENTS This work was funded by the Natural Science Foundation of China (81873972, 81873980, and 32172296) , the Foundation for Innovative Research Groups of Chongqing Higher Education Institutions (CXQT20013) , the Chongqing Science Fund for Distinguished Young Scholars (cstc2019jcyjjqX0028) , and the Chongqing Talents-Innova-tion Leading Talents Project (CQYC20200303107) ."