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Angew Chem Int Ed Engl. 2019 Apr 8;58(16):5382-5386. doi: 10.1002/anie.201901292. Epub 2019 Mar 12.

An RNA-Guided Cas9 Nickase-Based Method for Universal Isothermal DNA Amplification.

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Lab of Biosystem and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science & Technology, Shanghai, 200237, China.
Institute of Engineering Biology and Health, Collaborative Innovation Center of Yangtze River Delta Region Green Pharmaceuticals, College of Pharmaceutical Sciences, Zhejiang University of Technology, Hangzhou, 310014, Zhejiang, China.
School of Chemistry and Chemical Engineering, Shihezi University, Xinjiang, 832000, China.


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 °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 μL 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.


Cas9 nickase; DNA detection; isothermal amplification; single-guide RNA


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