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

Zhou, Wenhua; Li, Hu; Ying, Liming; Zhao, Zhen; Chu, Paul K.; Yu, Xue‐Feng

doi:10.1038/s41467-018-07324-5

Cited by 278 publications

(193 citation statements)

References 51 publications

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“…Compared to previously reported CRISPR-based nucleic acid detection, 14,16,18,23,24,25 our versatile and robust AIOD-CRISPR has some distinctive advantages. First, AIOD-CRISPR system is a true single reaction system.…”

Section: Discussionmentioning

confidence: 96%

All-in-One Dual CRISPR-Cas12a (AIOD-CRISPR) Assay: A Case for Rapid, Ultrasensitive and Visual Detection of Novel Coronavirus SARS-CoV-2 and HIV virus

Ding

Yin

et al. 2020

Preprint

119

129

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A recent outbreak of novel coronavirus (SARS-CoV-2), the causative agent of COVID-19, has spread rapidly all over the world. Human immunodeficiency virus (HIV) is another deadly virus and causes acquired immunodeficiency syndrome (AIDS). Rapid and early detection of these viruses will facilitate early intervention and reduce disease transmission risk. Here, we present an All-In-One Dual CRISPR-Cas12a (termed "AIOD-CRISPR") assay method for simple, rapid, ultrasensitive, one-pot, and visual detection of coronavirus SARS-CoV-2 and HIV virus. In our AIOD CRISPR assay, a pair of crRNAs was introduced to initiate dual CRISPR-Cas12a detection and improve detection sensitivity. The AIOD-CRISPR assay system was successfully utilized to detect nucleic acids (DNA and RNA) of SARS-CoV-2 and HIV with a sensitivity of few copies. Also, it was evaluated by detecting HIV-1 RNA extracted from human plasma samples, achieving a comparable sensitivity with real-time RT-PCR method. Thus, our method has a great potential for developing next-generation point-of-care molecular diagnostics.

show abstract

Section: Discussionmentioning

confidence: 96%

All-in-One Dual CRISPR-Cas12a (AIOD-CRISPR) Assay: A Case for Rapid, Ultrasensitive and Visual Detection of Novel Coronavirus SARS-CoV-2 and HIV virus

Ding

Yin

et al. 2020

Preprint

119

129

View full text Add to dashboard Cite

show abstract

“…Currently, our approach can achieve a detection limit of ~0.5 nM without any target amplification. To improve the sensitivity, our assay can be easily integrated with established isothermal nucleic acids amplification methods such as recombinase polymerase amplification (RPA) 31,15 or Loop-mediated isothermal amplification (LAMP) 32,33 to extend the detection limit to attomolar level. The amplification reagents can be mixed with CRISPR Cas12a protein and crRNA as a "one-pot" assay.…”

Section: Discussionmentioning

confidence: 99%

Magnetic Bead-Quantum Dot (MB-Qdot) CRISPR Assay for Instrument-Free Viral DNA Detection

Bao

Jensen

Chang

et al. 2020

Preprint

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We have developed a novel detection system which couples CRISPR-Cas recognition of target sequences, Cas mediated nucleic acid probe cleavage, and quantum dots as highly sensitive reporter molecules for instrument-free detection of viral nucleic acid targets. After target recognition and Cas mediated cleavage of biotinylated ssDNA probe molecules, the probe molecules are bound to magnetic particles. A complimentary ssDNA oligonucleotide quantum dot conjugate is then added, which only hybridizes to un-cleaved probes on the magnetic beads. After separation of hybridized from unhybridized quantum dot conjugates by magnetic sequestration, the signal is measured fluorometrically to provide a signal proportional to the cleaved probes and therefore the amount of target nucleic acid. To demonstrate the power of this assay, a 250 bp DNA target sequence matching a portion of the African swine fever virus (ASFV) genome is used and a detection limit of ~0.5 nM is achieved without target amplification using a simple portable ultraviolet flashlight. The positive samples are readily confirmed by visual inspection, completely avoiding the need for complicated devices and instruments. This work establishes the feasibility of a simple, instrument free assay for rapid nucleic acid screening in both hospitals and pointof-care settings. KEYWORDS -CRISRPR-Cas12a, quantum dot (Qdot), magnetic bead (MB), point-of-care (POC), pathogen detection was not certified by peer review) designed the experiments. M. Bao, Y. Chang, and G. Korensky conducted the experiments. M. Bao and K. Du wrote the manuscript. All authors commented the manuscript.

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“…For example, Cas9 nuclease specifically cleaves the dsDNA or ssDNA target to produce a fragment as a template or a primer for EXPAR or SDA . Yu and co‐workers developed a CRISPR‐Cas9‐triggered nicking endonuclease‐mediated SDA method (CRISDA) using a peptide nucleic acid (PNA) invasion‐mediated endpoint measurement . CRISDA exhibited ultrahigh sensitivity and single‐base specificity, however, it also required complex reaction reagents, including spyCas9‐sgRNA, nicking endonuclease, single‐stranded DNA binding protein (SSB), a Klenow fragment, a primer pair, a biotin‐labeled PNA probe, a Cy5‐labeled PNA probe, and streptavidin‐labeled magnetic beads.…”

Section: Methodsmentioning

confidence: 99%

An RNA‐Guided Cas9 Nickase‐Based Method for Universal Isothermal DNA Amplification

et al. 2019

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We have developed an ingenious method, termed Cas9 nickase‐based amplification reaction (Cas9nAR), to amplify a target fragment from genomic DNA at a constant temperature of 37 °C. Cas9nAR employs a sgRNA:Cas9n complex with a single‐strand nicking property, a strand‐displacing DNA polymerase, and two primers bearing the cleavage sequence of Cas9n, to promote cycles of DNA replication through priming, extension, nicking, and displacement reaction steps. Cas9nAR exhibits a zeptomolar limit of detection (2 copies in 20 μL of reaction system) within 60 min and a single‐base discrimination capability. More importantly, the underlying principle of Cas9nAR offers simplicity in primer design and universality in application. Considering the superior sensitivity and specificity, as well as the simple‐to‐implement, rapid, and isothermal features, Cas9nAR holds great potential to become a routine assay for the quantitative detection of nucleic acids in basic and applied studies.

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

Cited by 278 publications

References 51 publications

All-in-One Dual CRISPR-Cas12a (AIOD-CRISPR) Assay: A Case for Rapid, Ultrasensitive and Visual Detection of Novel Coronavirus SARS-CoV-2 and HIV virus

All-in-One Dual CRISPR-Cas12a (AIOD-CRISPR) Assay: A Case for Rapid, Ultrasensitive and Visual Detection of Novel Coronavirus SARS-CoV-2 and HIV virus

Magnetic Bead-Quantum Dot (MB-Qdot) CRISPR Assay for Instrument-Free Viral DNA Detection

An RNA‐Guided Cas9 Nickase‐Based Method for Universal Isothermal DNA Amplification

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