A novel method to detect mutation in DNA by utilizing exponential amplification reaction triggered by the CRISPR-Cas9 system

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We, herein, describe a novel method to detect mutation in DNA by utilizing exponential amplification reaction (EXPAR) triggered by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9, called CRISPR-EXPAR. The CRISPR system consisting of two Cas9/sgRNA complexes was designed to cut out a specific mutation region within the target DNA, which would consequently promote EXPAR by continuously repeated extension and nicking reactions. As a consequence, a large number of final EXPAR products, which can be monitored through duplex-specific fluorescent staining, are produced. Based on this design principle, we successfully identified a model target mutation within the human epidermal growth factor receptor 2 (HER2) gene down to 437 aM with excellent specificity. The practical capability of this method was verified by reliably identifying the target mutation directly from the genomic DNA (gDNA) extracted from the lung cancer cell line, NCI-H1781 (H1781), and its universal applicability was further confirmed by identifying another EFGF L858R mutation. This technique could serve as a new isothermal platform to identify various mutations by rationally redesigning single guide RNA (sgRNA) according to the target mutation site.
Publisher
ROYAL SOC CHEMISTRY
Issue Date
2021-04
Language
English
Article Type
Article
Citation

NANOSCALE, v.13, no.15, pp.7193 - 7201

ISSN
2040-3364
DOI
10.1039/d1nr00438g
URI
http://hdl.handle.net/10203/285311
Appears in Collection
CBE-Journal Papers(저널논문)
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