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

• DOI: 10.1038/s41467-018-07324-5
• Publication Date: 2018-11-21T14:47:37Z
• Authors: Wenhua Zhou; Li Hu; Liming Ying; Zhen Zhao; Paul K. Chu; Xue-Feng Yu
• Keywords: 570; Science; 610; Article; Fluorescence; 03 medical and health sciences; CRISPR/CAS; CRISPR-Associated Protein 9; NUCLEIC-ACID DETECTION; MD Multidisciplinary; Humans; CAS9; SPECIFICITY; T4 GENE-32 PROTEIN; 0303 health sciences; Science & Technology; Base Sequence; Nucleotides; TARGETED ENRICHMENT; Q; RECOGNITION; DNA; Multidisciplinary Sciences; HEK293 Cells; SINGLE; INTERROGATION; Science & Technology - Other Topics; RNA; CRISPR-Cas Systems; Nucleic Acid Amplification Techniques

AbstractAlthough 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.