Controllable crRNA Self-Transcription Aided Dual-Amplified CRISPR-Cas12a Strategy for Highly Sensitive Biosensing of FEN1 Activity

作者全名："Song, Yang; Gao, Ke; Cai, Xiaoying; Cheng, Wei; Ding, Shijia; Zhang, Decai; Deng, Shixiong"

作者地址："[Gao, Ke; Cai, Xiaoying; Cheng, Wei; Ding, Shijia; Deng, Shixiong] Chongqing Med Univ, Coll Basic Med, Lab Forens Med & Biomed Informat, Chongqing 400016, Peoples R China; [Song, Yang] Army Med Univ, Mil Med Univ 3, Daping Hosp, Inst Surg Res,Canc Ctr, Chongqing, Peoples R China; [Gao, Ke; Cai, Xiaoying; Ding, Shijia] Chongqing Med Univ, Coll Lab Med, Key Lab Clin Lab Diagnost, Ministryof Educ, Chongqing 400016, Peoples R China; [Cheng, Wei] Chongqing Med Univ, Affiliated Hosp 1, Ctr Clin Mol Med Detect, Chongqing 400016, Peoples R China; [Zhang, Decai] Shenzhen Univ, Shenzhen Univ, Affiliated Hosp 3, Dept Lab Diag, Shenzhen 518000, Peoples R China"

通信作者："Deng, SX (通讯作者)，Chongqing Med Univ, Coll Basic Med, Lab Forens Med & Biomed Informat, Chongqing 400016, Peoples R China.; Zhang, DC (通讯作者)，Shenzhen Univ, Shenzhen Univ, Affiliated Hosp 3, Dept Lab Diag, Shenzhen 518000, Peoples R China."

来源：ACS SYNTHETIC BIOLOGY

ESI学科分类：BIOLOGY & BIOCHEMISTRY

WOS号：WOS:000873777700001

JCR分区：Q1

影响因子：4.7

年份：2022

卷号：　

期号：　

开始页：　

结束页：　

文献类型：Article; Early Access

关键词：CRISPR/Cas12a; trans-cleavage; crRNA; FEN1; biosensor

摘要："A controllable crRNA self-transcription aided dual-amplified CRISPR-Cas12a strategy (termed CST-Cas12a) was developed for highly sensitive and specific biosensing of flap endonuclease 1 (FEN1), a structure-selective nuclease in eukaryotic cells. In this strategy, a branched DNA probe with a 5' overhanging flap was designed to serve as a hydrolysis substrate of FEN1. The flap cut by FEN1 was annealed with a template probe and functioned as a primer for an extension reaction to produce a double-stranded DNA (dsDNA) containing a T7 promoter and crRNA transcription template. Assisting the T7 RNA polymerase, abundant crRNA was generated and assembled with Cas12a to form a Cas12a/crRNA complex, which can be activated by a dsDNA trigger and unlock the indiscriminate fluorophore-quencher reporter cleavage. The highly efficient dual signal amplification and near-zero background enabled CST-Cas12a with extraordinarily high sensitivity. Under optimized conditions, this method allowed highly sensitive biosensing of FEN1 activity in the range of 1 x 10(-5) U mu L-1 to 5 x 10(-2) U mu L-1 with a detection limit of 5.2 x 10(-6) U mu L-1 and achieved excellent specificity for FEN1 in the presence of other interfering enzymes. The inhibitory capabilities of chemicals on FEN1 were also investigated. Further, the newly established CST-Cas12a strategy was successfully applied to FEN1 biosensing in complex biological samples, which might be a reliable biosensing platform for highly sensitive and specific detection of FENI activity in clinical applications."

基金机构：National Natural Science Foundation of China; Natural Science Foundation of Chongqing; [81873980]; [82102505]; [cstc2021jcyj-msxmX0485]

基金资助正文："? ACKNOWLEDGMENTS This work was funded by financial support from the National Natural Science Foundation of China (81873980, 82102505) and the Natural Science Foundation of Chongqing (cstc2021jcyj-msxmX0485) ."