Sensitive and Portable Signal Readout Strategies Boost Point-of-Care CRISPR/Cas12a Biosensors

Li, Tong; Cheng, Nan

doi:10.1021/acssensors.3c01338

Cited by 5 publications

(3 citation statements)

References 121 publications

(260 reference statements)

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“…[16][17][18] This technology is regarded as a powerful and immensely promising tool for nucleic acid detection, potentially surpassing qPCR technology to become the new gold standard. [19][20][21][22] DETECTR 13 and HOLMES 16 , developed in 2018 using CRISPR-Cas12a, marked the beginning of a period of rapid development for the CRISPR-Cas12a nucleic acid detection system. Typically, CRISPR-Cas12abased nucleic acid detection systems require speci c crRNA design, including a universal direct repeat (DR) region and a spacer region (18-23 nt).…”

Section: Introductionmentioning

confidence: 99%

“…[30][31][32] The CRISPR-Cas12a system holds great potential in the eld of clinical nucleic acid testing, offering improved detection sensitivity, reduced reliance on large instruments, lower testing costs, and faster detection speed. [19][20][21] It allows for early screening of low copy number viruses and tumor nucleic acid biomarkers. 29,33,34 However, integrating the isothermal ampli cation system with the CRISPR reaction in a single tube can hinder nucleic acid ampli cation e cacy due to the trans-cleavage activity of the Cas12a protein, particularly with low copy number inputs.…”

Section: Introductionmentioning

confidence: 99%

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Enhancement of CRISPR-Cas12a System through Universal Circular RNA Design

Liu,

Wang,

Zhang

et al. 2024

Preprint

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The combination of isothermal amplification and CRISPR-Cas12a system is a valuable tool for detecting nucleic acids, especially in emergency situations or disease outbreaks. However, achieving compatibility between these technologies has been a challenge because the CRISPR reaction eagerly cleaves nucleic acid amplification template before isothermal amplification is complete. This study presents a method called CIRCLE, which effectively addresses the compatibility issue by dividing crRNA into a universal circular DR region with a PC linker and a replaceable spacer region. Different targets could be detected in a single separate way by changing the spacer region. Furthermore, DNA modifications were firstly introduced into circular crRNA to regulate CRISPR-Cas12a system using base excision repair (BER) enzymes. The programmable strategy showed potential to detect different BER enzymes and regulate Cas12a gene-editing activity by adjusting BER enzyme levels. In the future, it holds promise for selectively editing genes in cells that express high levels of specific BER enzymes, such as UDG, in colon cancer cells. The findings of this study contribute to advancing the field of nucleic acid detection and gene editing. The CIRCLE method is a programmable and resource efficient approach for medical diagnostic applications, making it particularly valuable in future clinical settings.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Enhancement of CRISPR-Cas12a System through Universal Circular RNA Design

Liu,

Wang,

Zhang

et al. 2024

Preprint

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“…Proximity-based assays, such as proximity extension assays (PEA), transduce the molecular protein-binding events to oligonucleotide hybridization and downstream signal generation via isothermal amplification, − opening the possibility of exploiting nucleic acid-based signal amplification for protein biomarker detection. Cas12a-based protein detection methods have also emerged in recent years for sensitive detection. − While both PEA and Cas12a-based approaches have the advantage of being highly sensitive, their workflow typically includes multiple steps, leading to high assay complexity and long reaction times. In this study, we introduced a Cas12a-based isothermal homogeneous immunoassay for protein detection in a one-pot format.…”

mentioning

confidence: 99%

One-Pot Detection of Proteins Using a Two-Way Extension-Based Assay with Cas12a

Gao,

Ang,

Yung

2024

ACS Sens.

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Protein biomarkers are an important class of biomarkers in disease diagnosis and are traditionally detected by enzyme-linked immunosorbent assay and mass spectrometry, which involve multiple steps and a complex workflow. In recent years, many CRISPR-Cas12a-based methods for protein detection have been developed; however, most of them have not overcome the workflow complications observed in traditional assays, limiting their applicability in point-of-care testing. In this work, we designed a single-step, one-pot, and proximity-based isothermal immunoassay integrating CRISPR Cas12a for homogeneous protein target detection with a simplified workflow and high sensitivity. Probes consisting of different binders (small molecule, aptamer, and antibody) conjugated with oligonucleotides undergo two-way extension upon binding to the protein targets, leading to downstream DNA amplification by a pair of nicking enzymes and polymerases to generate target sequences for Cas12a signal generation. We used the streptavidin−biotin model to demonstrate the design of our assay and proved that all three elements of protein detection (target protein binding, DNA amplification, and Cas12a signal generation) could coexist in one pot and proceed isothermally in a single buffer system at a low reaction volume of 10 μL. The plug-and-play applicability of our assay has been successfully demonstrated using four different protein targets, streptavidin, PDGF-BB, antidigoxigenin antibody, and IFNγ, with the limit of detection ranging from fM to pM.

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Freestanding Nanofiber‐Assembled Aptasensor for Precisely and Ultrafast Electrochemical Detection of Alzheimer's Disease Biomarkers

Liu,

Yuan,

Liu

et al. 2024

Adv Healthcare Materials

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Amyloid beta‐protein (Aβ) is a main hallmark of Alzheimer's disease (AD), and a low amount of Aβ protein accumulation appears to be a potential marker for AD. Here, we developed an electrochemical DNA biosensor based on polyamide/polyaniline carbon nanotubes (PA/PANI‐CNTs) with the aim of diagnosing Alzheimer's disease early using a simple, low‐cost, and accessible method to rapidly detect Aβ42 in human blood. Electrospun PA nanofibers served as the skeleton for the successive in‐situ deposition of PANI and CNTs, which contributed both high conductivity and abundant binding sites for the Aβ42 aptamers. After the aptamers were immobilized, this aptasensor exhibited precise and specific detection of Aβ42 in human blood within only 4 min with an extremely fastest response rate, lower detection limit, and excellent linear detection range. These findings make a significant contribution to advancing the development of serum‐based detection techniques for Aβ42, thereby paving the way for improved diagnostic capabilities in the field of AD.This article is protected by copyright. All rights reserved

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Sensitive and Portable Signal Readout Strategies Boost Point-of-Care CRISPR/Cas12a Biosensors

Cited by 5 publications

References 121 publications

Enhancement of CRISPR-Cas12a System through Universal Circular RNA Design

Enhancement of CRISPR-Cas12a System through Universal Circular RNA Design

One-Pot Detection of Proteins Using a Two-Way Extension-Based Assay with Cas12a

Freestanding Nanofiber‐Assembled Aptasensor for Precisely and Ultrafast Electrochemical Detection of Alzheimer's Disease Biomarkers

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