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.
Highlights
Polymerase chain reaction (PCR) is the most widely used method for DNA amplification, the requirement of thermocycling limits its non-laboratory applications
Owing to the limitation of power-hungry thermocycling in conventional polymerase chain reactions (PCRs), efficient isothermal DNA amplification tools are of great importance to basic research as well as diagnostic/monitoring applications
Several methods such as the padlock/molecular inversion probes (MIP)-mediated methods, nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), and recombinase polymerase amplification (RPA) have been proposed[1,2,3,4,5,6,7], they suffer from trade-offs with regard to sensitivity, specificity, simplicity, and cost
Summary
Polymerase chain reaction (PCR) is the most widely used method for DNA amplification, the requirement of thermocycling limits its non-laboratory applications. Owing to the limitation of power-hungry thermocycling in conventional polymerase chain reactions (PCRs), efficient isothermal DNA amplification tools are of great importance to basic research as well as diagnostic/monitoring applications Several methods such as the padlock/molecular inversion probes (MIP)-mediated methods, nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), and recombinase polymerase amplification (RPA) have been proposed[1,2,3,4,5,6,7], they suffer from trade-offs with regard to sensitivity, specificity, simplicity, and cost. CRISDA is demonstrated to be a powerful tool in ultrasensitive detection and diagnosis of nucleic acids
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