Integrating CRISPR-Cas12a with a crRNA-Mediated Catalytic Network for the Development of a Modular and Sensitive Aptasensor

作者全名："Shu, Xiaojia; Zhang, Decai; Li, Xingrong; Zheng, Qingyuan; Cai, Xiaoying; Ding, Shijia; Yan, Yurong"

作者地址："[Shu, Xiaojia; Li, Xingrong; Zheng, Qingyuan; Cai, Xiaoying; Ding, Shijia; Yan, Yurong] Chongqing Med Univ, Coll Lab Med, Key Lab Clin Lab Diagnost, Minist Educ, Chongqing 400016, Peoples R China; [Zhang, Decai] Shenzhen Univ, Affiliated Hosp 3, Dept Lab Diag, Shenzhen 518000, Peoples R China"

通信作者："Yan, YR (通讯作者)，Chongqing Med Univ, Coll Lab Med, Key Lab Clin Lab Diagnost, Minist Educ, Chongqing 400016, Peoples R China."

来源：ACS SYNTHETIC BIOLOGY

ESI学科分类：BIOLOGY & BIOCHEMISTRY

WOS号：WOS:000841094200001

JCR分区：Q1

影响因子：3.7

年份：2023

卷号：　

期号：　

开始页：　

结束页：　

文献类型：Article; Early Access

关键词：aptasensor; CRISPR RNA; Cas12a; molecular network; ATP

摘要："Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a, which exhibits excellent target DNA-activated trans-cleavage activity under the guidance of a programmable CRISPR RNA (crRNA), has shown great promise in next-generation biosensing technology. However, current CRISPR-Cas12a-based biosensors usually improve sensitivity by the initial nucleic acid amplification, while the distinct programmability and predictability of the crRNA-guided target binding process has not been fully exploited. Herein, we, for the first time, propose a modular and sensitive CRISPR-Cas12a fluorometric aptasensor by integrating an enzyme-free and robust crRNA-mediated catalytic nucleic acid network, namely, Cas12a-CMCAN, in which crRNA acts as an initiator to actuate cascade toehold-mediated strand displacement reactions (TM-SDRs). As a proof of concept, adenosine triphosphate (ATP) was selected as a model target. Owing to the multiturnover of CRISPR-Cas12a trans-cleavage and the inherent recycling amplification network, this method achieved a limit of detection value of 0.16 mu M (20-fold lower than direct Cas12a-based ATP detection) with a linear range from 0.30 to 175 mu M. In addition, Cas12a-CMCAN can be successfully employed to detect ATP levels in diluted human serum samples. Considering the simplicity, sensitivity, and easy to tune many targets by changing aptamer sequences, the Cas12a-CMCAN sensing method is expected to offer a heuristic idea for the development of CRISPR-Cas12a-based biosensors and unlock its potential for general and convenient molecule diagnostics."

基金机构："National Natural Science Foundation of China [81371904, 81873980]; National Science and Technology Major Project of the Ministry of Science and Technology of China [2018ZX10732202]; Natural Science Foundation Project of Chongqing [cstc2018jcyjAX0349]"

基金资助正文："? ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (81371904, 81873980) , the National Science and Technology Major Project of the Ministry of Science and Technology of China (2018ZX10732202) , and the Natural Science Foundation Project of Chongqing (cstc2018jcyjAX0349) ."