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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.

Author information

1
Lab of Biosystem and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science & Technology, Shanghai, 200237, China.
2
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.
3
School of Chemistry and Chemical Engineering, Shihezi University, Xinjiang, 832000, China.

Abstract

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.

KEYWORDS:

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

PMID:
30773764
DOI:
10.1002/anie.201901292

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