文献类型：期刊文献

英文题名：An RNA-Guided Cas9 Nickase-Based Method for Universal Isothermal DNA Amplification

作者：Wang, Ting[1];Liu, Yong[1];Sun, Huan-Huan[1];Yin, Bin-Cheng[1];Ye, Bang-Ce[1,2,3]

机构：[1]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, Lab Biosyst & Microanal, Shanghai 200237, Peoples R China;[2]Zhejiang Univ Technol, Coll Pharmaceut Sci, Collaborat Innovat Ctr Yangtze River Delta Reg Gr, Inst Engn Biol & Hlth, Hangzhou 310014, Zhejiang, Peoples R China;[3]Shihezi Univ, Sch Chem & Chem Engn, Xinjiang 832000, Peoples R China

年份：2019

卷号：58

期号：16

起止页码：5382

外文期刊名：ANGEWANDTE CHEMIE-INTERNATIONAL EDITION

收录：;EI(收录号：20191106627763);WOS:【SCI-EXPANDED(收录号:WOS:000463975300037)】；

基金：This work was jointly supported by the National Natural Science Foundation of China (Grants 21822402, 21421004, 21675052, 21335003, 21575089), the Fundamental Research Funds for the Central Universities, and Programme of Introducing Talents of Discipline to University (Grants B18022, B16017).

语种：英文

外文关键词：Cas9 nickase; DNA detection; isothermal amplification; single-guide RNA

摘要：We have developed an ingenious method, termed Cas9 nickase-based amplification reaction (Cas9nAR), to amplify a target fragment from genomic DNA at a constant temperature of 37 degrees C Cas9nAR employs a sgRNA:Cas9n complex with a single-strand nicking property, a strand-displacing DNA polymerase, and two primers bearing the cleavage sequence of Cas9n, to promote cycles of DNA replication through priming, extension, nicking, and displacement reaction steps. Cas9nAR exhibits a zeptomolar limit of detection (2 copies in 20 mu d, of reaction system) within 60 min and a single-base discrimination capability. More importantly, the underlying principle of Cas9nAR offers simplicity in primer design and universality in application. Considering the superior sensitivity and specificity, as well as the simple-to-implement, rapid, and isothermal features, Cas9nAR holds great potential to become a routine assay for the quantitative detection of nucleic acids in basic and applied studies.