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

Ting WangYong LiuHuan-Huan SunBin-Cheng YinBang-Ce Ye
Published in: Angewandte Chemie (International ed. in English) (2019)
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.
Keyphrases
  • crispr cas
  • genome editing
  • nucleic acid
  • circulating tumor
  • cell free
  • gene expression
  • label free
  • risk assessment
  • copy number
  • clinical practice
  • genome wide
  • circulating tumor cells