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

• 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
• ISSN: 1433-7851; 1521-3773
• DOI: 10.1002/anie.201901292

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.