Trends in developing one-pot CRISPR diagnostics strategies

Trends in biotechnology, Год журнала: 2024, Номер unknown

Опубликована: Авг. 1, 2024

Язык: Английский

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FRAME: flap endonuclease 1-engineered PAM module for precise and sensitive modulation of CRISPR/Cas12a trans-cleavage activity

Nucleic Acids Research, Год журнала: 2024, Номер 52(19), С. 11884 - 11894

Опубликована: Сен. 24, 2024

Abstract CRISPR/Cas12a system, renowned for its precise recognition and efficient nucleic acid cleavage capabilities, has demonstrated remarkable performance in molecular diagnostics biosensing. However, the reported Cas12a activity regulation methods often involved intricate CRISPR RNA (crRNA) structural adjustments or costly chemical modifications, which limited their applications. Here, we a unique enzyme engineering strategy using flap endonuclease 1 (FEN1) to regulate accessibility of protospacer adjacent motif (PAM) module double-stranded DNA activator (FRAME). By identifying three-base overlapping structure between target inputs substrate, FEN1 selectively cleaved released 5′-flap containing ‘TTTN’ sequence, triggered secondary while forming nicked PAM, ultimately achieving sensitive switching Cas12a’s activity. The FRAME exemplified ‘two birds with one stone’ principle, as it not only precisely programmed but also simultaneously isothermal cyclic amplification. Moreover, was applied construct sensing platform detecting myeloperoxidase miR-155, high sensitivity specificity. Importantly, proved versatility multiple targets single crRNA without redesign. Collectively, opens up novel avenue modulating activity, promising immense potential realm medical diagnostics.

Язык: Английский

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DNAzyme-Triggered Equilibrium Transfer with Self-Activated CRISPR-Cas12a Biosensor Enables One-Pot Diagnosis of Nucleic Acids

Analytical Chemistry, Год журнала: 2025, Номер 97(5), С. 3026 - 3035

Опубликована: Янв. 31, 2025

Integrating recombinase-polymerase amplification (RPA) with CRISPR-Cas12a holds significant potential to simplify and improve nucleic acid diagnostic procedures. However, current strategies face limitations, such as complexity, reduced efficiency, compromises in Cas12a activity. In response, we developed a DNAzyme-triggered equilibrium transfer self-activated biosensor (DESCRIBER) for integrated detection. This platform features varying balance points minimize interference between RPA one pot maximize their activity at different stages. Initially, the reaction focused on RPA, while was silenced by circular-crRNA (C-crRNA). Then, DNAzyme, activator, generated during process, which linearizes C-crRNA activate toward signal readout. Meanwhile, activated can further linearize promote self-activation accelerate transfer. According this principle, highly sensitive detection of HIV-1 genome, low 500 CPs/mL, achieved within 1 h maintaining universality detecting common subtypes specificity against opportunistic infectious pathogens. Compared qRT-PCR, it also exhibited good accuracy 35 spiked samples. Overall, believe that proposed strategy will enhance existing CRISPR systems practical applications clinical diagnosis.

Язык: Английский

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Engineered CRISPR/Cas Ribonucleoproteins for Enhanced Biosensing and Bioimaging

Analytical Chemistry, Год журнала: 2025, Номер unknown

Опубликована: Март 11, 2025

CRISPR-Cas systems represent a highly programmable and precise nucleic acid-targeting platform, which has been strategically engineered as versatile toolkit for biosensing bioimaging applications. Nevertheless, their analytical performance is constrained by inherent functional activity limitations of natural CRISPR/Cas systems, underscoring the critical role molecular engineering in enhancing capabilities. This review comprehensively examines recent advancements ribonucleoproteins (RNPs) to enhance capabilities advanced detection cellular imaging. We explore innovative strategies developing enhanced RNPs, including Cas protein through mutagenesis fusion techniques, guide RNA via chemical structural modifications. Furthermore, we evaluate these RNPs' applications sensitive biomarker live-cell genomic DNA monitoring, while analyzing current challenges prospective developments RNP bioimaging.

Язык: Английский

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CRISPR/Cas12a Protein Switch Powered Label-Free Electrochemical Biosensor for Sensitive Viral Protease Detection

Analytical Chemistry, Год журнала: 2025, Номер unknown

Опубликована: Март 31, 2025

Viral proteases are critical molecular targets in viral pathogenesis, representing pivotal biomarkers for understanding infection mechanisms and developing antiviral therapeutics. This study introduces a label-free electrochemical biosensor that enables sensitive protease detection by integrating protease-responsive CRISPR/Cas protein switches (CasPSs) with hemin aptamer-functionalized interface. The biosensor's mechanism relies on protease-mediated proteolysis, which leads to the release of active Cas12a proteins from CasPSs generates amplified responses through continuous cleavage immobilized redox-active hemin/aptamer complexes. achieved specific hepatitis C virus NS3/4A sensing femtomolar sensitivity could be readily expanded other replacing CasPS module. feasibility this was demonstrated monitoring enterovirus 71 3C activities virus-infected cell samples different loads postinfection times. provides promising strategy CRISPR biosensing platforms, offering helpful analytical tool drug screening.

Язык: Английский

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BE-CATCH: Bioamplifier-Equipped CRISPR-Cas12a Transduction System Coupled with Commercial Pregnancy Test Strips to Harness Signal-on Point-of-Care Detection

Analytical Chemistry, Год журнала: 2025, Номер unknown

Опубликована: Апрель 15, 2025

Repurposing existing commercial diagnostic equipment to enable portable analysis of diverse targets is driving the development affordable point-of-care testing (POCT). Interestingly, we found that goat antimouse IgG could replace human chorionic gonadotropin (hCG) make T line pregnancy test strips (PTS) appear red color and accordingly synthesized a novel signal output probe, which eliminated intricate hCG covalent coupling steps, meet multiple needs expanded POCT. Given this, introduced separation-free universal POCT strategy termed bioamplifier-equipped CRISPR-Cas12a transduction system coupled with PTS harness signal-on detection (BE-CATCH). Specifically, target inputs were converted amplified by multiplied strand displacement amplification-based bioamplifier, thereby activating Cas12a's trans-cleavage activity. Then, activated Cas12a would cleave connector indiscriminately, ultimately kept probe in free state; thus, be translated into colorimetric on PTS. This not only provided boosted sensitivity specificity but also enhanced user-friendliness maintaining mode. We demonstrated versatility BE-CATCH through selectively detecting miR-155 flap endonuclease 1. its broad adaptability, provide an appealing option broaden application biomedical diagnostics.

Язык: Английский

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Three birds with one stone: a facile superparamagnetic aptasensor for selective capture, dual-mode detection and efficient elimination of methicillin-resistant Staphylococcus aureus

Chemical Engineering Journal, Год журнала: 2025, Номер unknown, С. 163703 - 163703

Опубликована: Май 1, 2025

Язык: Английский

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Internal-Driven DNA Nanowindmill: Multi-Fulcrum Mediated Amplified Rolling Nanomachine for Fast and Ultrasensitive Electrochemical Detection of Tau

Sensors and Actuators B Chemical, Год журнала: 2024, Номер unknown, С. 136797 - 136797

Опубликована: Окт. 1, 2024

Язык: Английский

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Synthetic Protein-to-DNA Input Exchange for Protease Activity Detection Using CRISPR-Cas12a

Analytical Chemistry, Год журнала: 2024, Номер 96(47), С. 18645 - 18654

Опубликована: Ноя. 14, 2024

We present a novel activity-based detection strategy for matrix metalloproteinase 2 (MMP2), critical cancer protease biomarker, leveraging mechanism responsive to the proteolytic activity of MMP2 and its integration with CRISPR-Cas12a-assisted signal amplification. designed chemical translator comprising two functional units─a peptide nucleic acid (PNA), fused together. The presents substrate MMP2, while PNA serves as output subsequent processing. This was immobilized on micrometer magnetic beads physical support an assay. incorporated into our design single-stranded DNA partially hybridized sequence bearing region complementary RNA guide CRISPR-Cas12a. target-induced nuclease Cas12a results in degradation FRET-labeled reporters amplified fluorescence signal, enabling low picomolar range, showing limit 72 pg/mL. study provides new principles broader applicability CRISPR-Cas-based biosensing.

Язык: Английский

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Engineering stimuli-responsive CRISPR-Cas systems for versatile biosensing

Analytical and Bioanalytical Chemistry, Год журнала: 2024, Номер unknown

Опубликована: Ноя. 27, 2024

Язык: Английский

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