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

TLDR

PCR requires thermocycling, limiting its use outside laboratories, so isothermal amplification methods are valuable for on‑site diagnostics. The authors present CRISDA, a true isothermal method that amplifies and detects double‑stranded DNA using CRISPR–Cas9‑triggered nicking endonuclease‑mediated strand displacement amplification. CRISDA exploits CRISPR effector conformational changes for high sensitivity and specificity, couples a peptide nucleic acid invasion endpoint for attomolar detection and single‑nucleotide discrimination, and achieves sub‑attomolar sensitivity when combined with Cas9‑mediated target enrichment. CRISDA proves to be a powerful isothermal tool for ultrasensitive, specific nucleic acid detection suitable for point‑of‑care diagnostics and field analyses.

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.