Amplified detection of nucleic acids and proteins using an isothermal proximity CRISPR Cas12a assay

Li, Yongya; Mansour, Howaida; Watson, Colton J. F.; Tang, Yanan; MacNeil, Adam J.; Li, Feng

doi:10.1039/d0sc06113a

Cited by 55 publications

(42 citation statements)

References 27 publications

(40 reference statements)

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“…Furthermore, despite its simplicity, the design showed high binding affinity, and target specificity originating from bivalent binding and worked well even in crude cellular extracts. Recently, other, similar approaches have also used proximity-induced hybridization to produce advanced bivalent biosensors; these include an electrochemical proximity assay for the femtomolar detection of insulin [44], a biosensor employing nicking endonucleaseassisted isothermal fluorescence signal amplification for the detection of streptavidin or prostate-specific antigen (PSA) [45], a proximity hybridization-regulated DNA biogate for PSA detection [46], a DNAzyme-based DNA nanomotor for detection of thrombin [47], a cytosensor for the detection of circulating tumor cells [48], a scattering proximity immunoassay based on the dimerization of AuNPs for detecting carcinoembryonic antigen [49], and other enzymatic methods [50,51]. Moreover, proximity-induced hybridization has been combined with templated reaction techniques to identify antibodies [52].…”

Section: Biosensors Based On Proximity-induced Hybridizationmentioning

confidence: 99%

Biosensors Based on Bivalent and Multivalent Recognition by Nucleic Acid Scaffolds

et al. 2022

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Several biological macromolecules adopt bivalent or multivalent interactions to perform various cellular processes. In this regard, the development of molecular constructs presenting multiple ligands in a specific manner is becoming crucial for the understanding of multivalent interactions and for the detection of target macromolecules. Nucleic acids are attractive molecules to achieve this goal because they are capable of forming various, structurally well-defined 2D or 3D nanostructures and can bear multiple ligands on their structures with precisely controlled ligand–ligand distances. Thanks to the features of nucleic acids, researchers have proposed a wide range of bivalent and multivalent binding agents that strongly bind to target biomolecules; consequently, these findings have uncovered new biosensing strategies for biomolecule detection. To date, various bivalent and multivalent interactions of nucleic acid architectures have been applied to the design of biosensors with enhanced sensitivity and target accuracy. In this review, we describe not only basic biosensor designs but also recently designed biosensors operating through the bivalent and multivalent recognition of nucleic acid scaffolds. Based on these designs, strategies to transduce bi- or multivalent interaction signals into readable signals are discussed in detail, and the future prospects and challenges of the field of multivalence-based biosensors are explored.

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Section: Biosensors Based On Proximity-induced Hybridizationmentioning

confidence: 99%

Biosensors Based on Bivalent and Multivalent Recognition by Nucleic Acid Scaffolds

et al. 2022

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“…The isothermal proximity CRISPR Cas12a assay is a new CRISPR technique that recognises the target (protein or NA) based on proximity binding rather than crRNA recognition (iPCCA). Upon binding, it allows for primer extension, which results in the generation of a barcode (a pre-designed CRISPR targetable sequence) for signal amplification [ 69 ]. The proximity binding approach makes it possible to detect NNTs without PAM sequences.…”

Section: Advancements In Crispr Detectionmentioning

confidence: 99%

Next-Generation Diagnostic with CRISPR/Cas: Beyond Nucleic Acid Detection

Bhardwaj

Kant

Behera

et al. 2022

IJMS

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The early management, diagnosis, and treatment of emerging and re-emerging infections and the rising burden of non-communicable diseases (NCDs) are necessary. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas system has recently acquired popularity as a diagnostic tool due to its ability to target specific genes. It uses Cas enzymes and a guide RNA (gRNA) to cleave target DNA or RNA. The discovery of collateral cleavage in CRISPR-Cas effectors such as Cas12a and Cas13a was intensively repurposed for the development of instrument-free, sensitive, precise and rapid point-of-care diagnostics. CRISPR/Cas demonstrated proficiency in detecting non-nucleic acid targets including protein, analyte, and hormones other than nucleic acid. CRISPR/Cas effectors can provide multiple detections simultaneously. The present review highlights the technical challenges of integrating CRISPR/Cas technology into the onsite assessment of clinical and other specimens, along with current improvements in CRISPR bio-sensing for nucleic acid and non-nucleic acid targets. It also highlights the current applications of CRISPR/Cas technologies.

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“…Currently, CRISPR biosensors are no longer limited to nucleic acid analyses. The robust signal amplification mechanism of CRISPR-Cas12 and CRISPR-Cas13 makes it a promising tool for sensing non-nucleic-acid targets, such as pathogenic bacteria, exosomes, − small molecules, ,− proteins, − and ions . Unlike nucleic acid detection, which can achieve ultrahigh sensitivity through template amplification, there is currently no viable non-nucleic-acid target amplification method, so its detection sensitivity conventionally depends on the probe affinity and detector performance.…”

Section: Crispr-cas Systems For Crispr Diagnosticsmentioning

confidence: 99%

Advances in Clustered, Regularly Interspaced Short Palindromic Repeats (CRISPR)-Based Diagnostic Assays Assisted by Micro/Nanotechnologies

et al. 2021

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Clustered, regularly interspaced short palindromic repeats (CRISPR)-based diagnoses, derived from gene-editing technology, have been exploited for less than 5 years and are now reaching the stage of precommercial use. CRISPR tools have some notable features, such as recognition at physiological temperature, excellent specificity, and high-efficiency signal amplification capabilities. These characteristics are promising for the development of next-generation diagnostic technologies. In this Perspective, we present a detailed summary of which micro/nanotechnologies play roles in the advancement of CRISPR diagnosis and how they are involved. The use of nanoprobes, nanochips, and nanodevices, microfluidic technology, lateral flow strips, etc. in CRISPR detection systems has led to new opportunities for CRISPR-based diagnosis assay development, such as achieving equipment-free detection, providing more compact detection systems, and improving sensitivity and quantitative capabilities. Although tremendous progress has been made, CRISPR diagnosis has not yet reached its full potential. We discuss upcoming opportunities and improvements and how micro/nanotechnologies will continue to play key roles.

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Amplified detection of nucleic acids and proteins using an isothermal proximity CRISPR Cas12a assay

Abstract: Herein, we develop an isothermal proximity CRISPR Cas12a assay that harnesses the target-induced collateral cleavage activity of Cas12a for the quantitative profiling of gene expression and detection of proteins with high sensitivity and specificity.

Cited by 55 publications

References 27 publications

Biosensors Based on Bivalent and Multivalent Recognition by Nucleic Acid Scaffolds

Biosensors Based on Bivalent and Multivalent Recognition by Nucleic Acid Scaffolds

Next-Generation Diagnostic with CRISPR/Cas: Beyond Nucleic Acid Detection

Advances in Clustered, Regularly Interspaced Short Palindromic Repeats (CRISPR)-Based Diagnostic Assays Assisted by Micro/Nanotechnologies

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