Branched DNA switchable CRISPR-Cas12a system for sensing FEN1 activity

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

作者地址："[Li, Xingrong; Cai, Xiaoying; Shu, Xiaojia; Zeng, Zijie; Ding, Shijia; Yan, Yurong] Chongqing Med Univ, Coll Lab Med, Key Lab Clin Lab Diagnost, Minist Educ, Chongqing 400016, Peoples R China; [Zhang, Decai] Southern Med Univ, Guangdong Prov Peoples Hosp, Guangdong Acad Med Sci, Lab Med, Guangzhou 510000, 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：CHEMICAL ENGINEERING JOURNAL

ESI学科分类：ENGINEERING

WOS号：WOS:001026647700001

JCR分区：Q1

影响因子：13.3

年份：2023

卷号：470

期号：　

开始页：　

结束页：　

文献类型：Article

关键词：FEN1; CRISPR-Cas12a; Branched DNA; Steric hindrance effect; Biosensing; Intracellular imaging

摘要："FEN1 plays a crucial role in tumor progression and proliferation, and it has been considered a prospective cancer biomarker and druggable target. However, its detection is compromised by expenditure consumption and challenged by the lack of straightforward and high-efficiency strategies both in vitro and intracellular applica-tions. Herein, we describe an original technique for hypersensitive detection of FEN1 by incorporating structure-specific branched DNA (BD) blocked activator with CRISPR-Cas12a methodology. Given the presence of over-hanging structure at the 5 & PRIME; end region of the tripartite BD, it is difficult for the BD to activate Cas12a protein which could be ascribed to the larger steric hindrance effect at the 5 & PRIME; terminal regions and interferes with the approachability of the constituent elements for crRNA to fully hybridize with the complementary sequences of BD. However, the appearance of FEN1 can cleave the overhanging branch of BD to form an identifiable split DNA activator, thus successfully activating the cis/trans-cleavage activity of Cas12a. By introducing a fluorescence resonance energy transfer (FRET)-based single-stranded DNA reporter, consecutive degradation events lead to an immediately perceptible intensification in fluorescence intensity, realizing highly efficient detection of FEN1. The strategy has also been successfully applied to complex biological sample analysis and intracellular imaging, demonstrating its potential application in biochemical and molecular biology research as well as clinical diag-nosis. In addition, we preliminarily verified the feasibility of integrating the established strategy with an elec-trochemiluminescent (ECL) platform, and confirmed that this strategy can be further expanded to other miniaturized sensing devices and has great prospects for point-of-care applications."

基金机构："National Natural Science Foundation of China [81873980, 82102505]; Natural Science Foundation Project of Chongqing [cstc2018jcyjAX0349]"

基金资助正文："This work was supported by financial support from the National Natural Science Foundation of China [81873980, 82102505] , and the Natural Science Foundation Project of Chongqing [cstc2018jcyjAX0349] ."