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

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 tw...

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Bibliographic Details
Published in:Angewandte Chemie International Edition 2019-04, Vol.58 (16), p.5382-5386
Main Authors: Wang, Ting, Liu, Yong, Sun, Huan‐Huan, Yin, Bin‐Cheng, Ye, Bang‐Ce
Format: Article
Language:English
Subjects:
Amplification
Cas9 nickase
CRISPR-Associated Protein 9 - metabolism
CRISPR-Cas Systems
Deoxyribonucleic acid
DNA
DNA biosynthesis
DNA detection
DNA polymerase
DNA Replication
DNA, Bacterial - genetics
DNA-directed DNA polymerase
isothermal amplification
Nicking endonuclease
Nucleic Acid Amplification Techniques
Nucleic acids
Nucleotide sequence
Primers
Ribonucleic acid
RNA
RNA, Guide, CRISPR-Cas Systems
Salmonella typhimurium - genetics
single-guide RNA
Temperature
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Summary: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. In good nick: A method, termed Cas9 nickase‐based amplification reaction (Cas9nAR), for the isothermal amplification of a target fragment from genomic DNA was developed. The method has a zeptomolar limit of detection and a single‐nucleotide discrimination capability.
ISSN:1433-7851
1521-3773
DOI:10.1002/anie.201901292