Premium
An RNA‐Guided Cas9 Nickase‐Based Method for Universal Isothermal DNA Amplification
Author(s) -
Wang Ting,
Liu Yong,
Sun HuanHuan,
Yin BinCheng,
Ye BangCe
Publication year - 2019
Publication title -
angewandte chemie
Language(s) - English
Resource type - Journals
eISSN - 1521-3757
pISSN - 0044-8249
DOI - 10.1002/ange.201901292
Subject(s) - multiple displacement amplification , loop mediated isothermal amplification , cas9 , dna , biology , computational biology , primer extension , primer (cosmetics) , nucleic acid , crispr , polymerase chain reaction , microbiology and biotechnology , chemistry , genetics , base sequence , gene , dna extraction , organic chemistry
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.