MicroRNA Sensors Based on CRISPR/Cas12a Technologies: Evolution From Indirect to Direct Detection

Critical Reviews in Analytical Chemistry, Год журнала: 2024, Номер unknown, С. 1 - 17

Опубликована: Март 15, 2024

MicroRNA (miRNA) has emerged as a promising biomarker for disease diagnosis and potential therapeutic targets drug development. The detection of miRNA can serve noninvasive tool in diseases predicting prognosis. CRISPR/Cas12a system great nucleic acid due to its high sensitivity specificity, which been developed be versatile acid-based various fields. However, conversion from RNA DNA with or without amplification operation is necessary based on system, because dsDNA containing PAM sequence ssDNA traditionally considered the activator Cas12a. Until recently, direct by reported. In this review, we provide an overview evolution biosensors indirect direct, would beneficial development CRISPR/Cas12a-based sensors better performance miRNA.

Язык: Английский

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Construction of a self-priming hairpin probe for rapid and isothermally one-step detection of multiple long noncoding RNAs in living cells and tissues

Chemical Engineering Journal, Год журнала: 2025, Номер unknown, С. 159600 - 159600

Опубликована: Янв. 1, 2025

Язык: Английский

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Split crRNA Precisely Assisted Cas12a Expansion Strategy for Simultaneous, Discriminative, and Low-Threshold Determination of Two miRNAs Associated with Multiple Sclerosis

Analytical Chemistry, Год журнала: 2025, Номер 97(5), С. 2873 - 2882

Опубликована: Фев. 3, 2025

Multiple sclerosis (MS) can proceed into secondary progressive MS accompanied by persistent neurological deterioration; therefore, accurate diagnosis of is vital significance. Irregularities microRNAs (miRNAs) expression have been observed in MS, so miRNAs evaluated as novel biomarkers and therapeutic targets. Herein, a new strategy named split crRNA precisely assisted Cas12a expansion (SPACE) was developed for simultaneous, discriminative, low-threshold determination two MS-related miRNAs: miRNA-155 miRNA-326. On the one hand, owing to property that could activate Cas12a, were designed spacers combine with scaffold. These integrated crRNAs then recognized activators, activating enabling RNA target identification. other SPACE dexterously activator reporter probe, utilized Cas12a′s cis-cleavage achieve simultaneous detection differential signal output Moreover, trans-cleavage ultra-high efficiency assembled sensitive quantification total blood samples at low thresholds. Overall, diversified design enabled dual pot step, providing reliable tool clinical diagnosis.

Язык: Английский

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Nanotechnology-leveraged CRISPR/Cas systems: icebreaking in trace cancer-related nucleic acids biosensing

Molecular Cancer, Год журнала: 2025, Номер 24(1)

Опубликована: Март 14, 2025

As promising noninvasive biomarkers, nucleic acids provide great potential to innovate cancer early detection methods and promote subsequent diagnosis improve the survival rates of patient. Accurate, straightforward sensitive such acid-based biomarkers in complex biological samples holds significant clinical importance. However, low abundance creates huge challenges for their routine detection. next-generation diagnostic tool, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) with high programmability, sensitivity, fidelity, single-base resolution, precise acid positioning capabilities are extremely attractive trace (NABCBs), permitting rapid, ultra-sensitive specific More importantly, by combing nanotechnology, it can solve long-lasting problems poor accuracy simplicity, as well achieve integrated miniaturization portable point-of-care testing (POCT) existing literature lacks emphasis on this topic. Thus, we intend propose a timely one readers. This review will bridge gap providing insights CRISPR/Cas-based nano-biosensing development highlighting current state-of-art, challenges, prospects. We expect that better understanding valuable NABCBs detection, thereby facilitating advancements screening/detection/diagnostics win practical applications foreseeable future.

Язык: Английский

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RT-RPA-Assisted Scaffold RNA Transcription Amplification Activation Cas12a Trans-Cleavage Strategy for One-Pot miRNA Detection

Talanta, Год журнала: 2025, Номер unknown, С. 128049 - 128049

Опубликована: Март 1, 2025

Язык: Английский

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Dual Cas12a and multiplex crRNA CRISPR strategy ultrasensitive detection novel circRNA biomarker for the diagnosis of ovarian cancer

BMC Cancer, Год журнала: 2025, Номер 25(1)

Опубликована: Апрель 15, 2025

Язык: Английский

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CRISPR/Cas12a-Powered Amplification-Free RNA Diagnostics by Integrating T7 Exonuclease-Assisted Target Recycling and Split G-Quadruplex Catalytic Signal Output

Analytical Chemistry, Год журнала: 2024, Номер 96(25), С. 10451 - 10458

Опубликована: Июнь 11, 2024

Rapid and sensitive RNA detection is of great value in diverse areas, ranging from biomedical research to clinical diagnostics. Existing methods for often rely on reverse transcription (RT) DNA amplification or involve a time-consuming procedure poor sensitivity. Herein, we proposed CRISPR/Cas12a-enabled amplification-free assay rapid, specific, This assay, which termed T7/G4-CRISPR, involved the use T7-powered nucleic acid circuit convert single target into numerous activators via toehold-mediated strand displacement reaction T7 exonuclease-mediated recycling amplification, followed by activating Cas12a trans-cleavage linker strands inhibiting split G-Quadruplex (G4) assembly, thereby inducing fluorescence attenuation proportion input target. We first performed step-by-step validation entire process optimized parameters. Using optimal conditions, T7/G4-CRISPR was capable detecting as low 3.6 pM RNA, obtaining ∼100-fold improvement sensitivity compared with most direct assays. Meanwhile, its excellent specificity could discriminate nucleotide variants adjacent toehold region allow species-specific pathogen identification. Furthermore, applied it analyzing bacterial 16S rRNA 40 urine samples, exhibiting 90% 100% when validated RT-quantitative PCR. Therefore, envision that will serve promising sensing approach expand toolbox CRISPR-based

Язык: Английский

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Development and assessment of cutting-edge biotechnologies

Journal of Biosafety and Biosecurity, Год журнала: 2024, Номер 6(1), С. 51 - 63

Опубликована: Март 1, 2024

The emergence of advanced biotechnologies has intensified in recent years. rapid development these had a wide and profound impact globally, with the majority on frontier biosecurity technologies. global situation is currently highly challenging, characteristics internationalization, extremely serious harm, complex development. misuse abuse are common, which thereby endanger biosecurity. international community governments have attached great importance to cutting-edge implementing laws regulations control prevent biosecurity-related influences. By tracking progress new technologies generated from gene editing, drives, synthetic biology, related cross-disciplines as applied field, we analyzed trends their potential influence. On one hand, this paper proposes that there an urgent need for cooperation formulate management, strategies, measures jointly promote sound other should shoulder responsibility safeguarding restrict rational applications within legal ethical frameworks. This aims provide reference prevention support governance.

Язык: Английский

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Mini crRNA-Mediated CRISPR/Cas12a System Enhanced Imaging of Multiple MicroRNAs in Cells

Chemical & Biomedical Imaging, Год журнала: 2024, Номер unknown

Опубликована: Дек. 19, 2024

RNA imaging in live cells can provide comprehensive information on the expression, localization, degradation, storage, and regulation of cells, which is crucial for basic biology clinical research. Our previous research finds that slicing facilitated crRNA typical CRISPR/Cas12a system at a fitted site did not affect its trans-cleavage activity, was previously reported to be triggered by targeted ssDNA or dsDNA, mini crRNA-mediated (MCM-CRISPR/Cas12a) proposed. Here, we further apply it enhanced MicroRNAs designing activator as molecular beacon (MB), form hybrid double-stranded structure DNA/RNA with MicroRNA. When MicroRNA present, hairpin MB opened emits fluorescence. Simultaneously, DNA-RNA formed activates activity LbCas12a, partially cleaving single-stranded DNA extended from enhancing fluorescence intensity system. We designed MCM-CRISPR/Cas12a miRNA-21, miRNA-155, miRNA-10b successfully applied sensitive specific these both inside outside cells. This study provides new idea multiple within important studying distribution dynamic changes

Язык: Английский

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miR-Cabiner: A Universal microRNA Sensing Platform Based on Self-Stacking Cascaded Bicyclic DNA Circuit-Mediated CRISPR/Cas12a

Analytical Chemistry, Год журнала: 2024, Номер 97(1), С. 799 - 810

Опубликована: Дек. 20, 2024

CRISPR/Cas12a-based diagnostics have great potential for sensing nucleic acids, but their application is limited by the sequence-dependent property. A platform termed miR-Cabiner (a universal miRNA based on self-stacking cascaded bicyclic DNA circuit-mediated CRISPR/Cas12a) demonstrated herein that sensitive and analyzing miRNAs. This combines catalytic hairpin assembly (CHA) hybrid chain reaction (HCR) into a unified circuit finally cascades to CRISPR/Cas12a. Compared with CHA–Cas12a HCR–Cas12a systems, exhibits significantly higher rate. Panels of miRNAs (miR-130a, miR-10b, miR-21, miR-1285), which are associated diagnosis, staging, prognosis breast cancer, designed demonstrate universality miR-Cabiner. Four can be detected fM-level simply tuning sequence in CHA components. Additionally, panel analysis also shows high accuracy practical samples. universally applicable detecting may serve as an excellent tool clinical diagnosis.

Язык: Английский

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