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

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

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Associated Protein 9 / metabolism*
  • CRISPR-Cas Systems
  • DNA Replication
  • DNA, Bacterial / genetics*
  • Nucleic Acid Amplification Techniques*
  • RNA, Guide, CRISPR-Cas Systems
  • Salmonella typhimurium / genetics
  • Temperature*

Substances

  • DNA, Bacterial
  • CRISPR-Associated Protein 9