A CRISPR–Cas9-triggered strand displacement amplification method for ultrasensitive DNA detection

Wenhua Zhou, Li Hu, Liming Ying, Zhen Zhao, Paul K. Chu and Xue-Feng Yu ()
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Wenhua Zhou: Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences
Li Hu: Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences
Liming Ying: National Heart and Lung Institute, Imperial College London
Zhen Zhao: Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences
Paul K. Chu: City University of Hong Kong
Xue-Feng Yu: Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences

Nature Communications, 2018, vol. 9, issue 1, 1-11

Abstract: Abstract Although polymerase chain reaction (PCR) is the most widely used method for DNA amplification, the requirement of thermocycling limits its non-laboratory applications. Isothermal DNA amplification techniques are hence valuable for on-site diagnostic applications in place of traditional PCR. Here we describe a true isothermal approach for amplifying and detecting double-stranded DNA based on a CRISPR–Cas9-triggered nicking endonuclease-mediated Strand Displacement Amplification method (namely CRISDA). CRISDA takes advantage of the high sensitivity/specificity and unique conformational rearrangements of CRISPR effectors in recognizing the target DNA. In combination with a peptide nucleic acid (PNA) invasion-mediated endpoint measurement, the method exhibits attomolar sensitivity and single-nucleotide specificity in detection of various DNA targets under a complex sample background. Additionally, by integrating the technique with a Cas9-mediated target enrichment approach, CRISDA exhibits sub-attomolar sensitivity. In summary, CRISDA is a powerful isothermal tool for ultrasensitive and specific detection of nucleic acids in point-of-care diagnostics and field analyses.

Date: 2018
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DOI: 10.1038/s41467-018-07324-5

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