‘Splice-at-will’ Cas12a crRNA engineering enabled direct quantification of ultrashort RNAs

Nucleic Acids Research, Год журнала: 2025, Номер 53(2)

Опубликована: Янв. 11, 2025

Abstract We present a robust ‘splice-at-will’ CRISPR RNA (crRNA) engineering mechanism that overcomes the limitations of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system in directly detecting ultrashort RNAs. In this strategy, an intact Cas12a crRNA can be split from almost any site spacer region to obtain truncated (tcrRNA) cannot activate even after binding auxiliary DNA activator. While splicing tcrRNAs with moiety RNA, formed combination work together efficiently, enabling engineering. Importantly, exhibits same trans-cleavage activation efficiency as conventional crRNA. Therefore, by rationally designing activator conserved tcrRNA-complementary sequence and arbitrary RNA-of-interest recognition domain, general sensing is established utilizes traditional DNA-activated detect This strategy could faithfully sequences 6–8 nt, which achieved Cas13a systems. Additionally, through flexible design, our method precisely distinguish single-base differences microRNA other sequences. has significantly expanded Cas12a-based diagnostic toolbox opened new avenues for detection.

Язык: Английский

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Programmable DNA Nanoswitch-Regulated Plasmonic CRISPR/Cas12a-Gold Nanostars Reporter Platform for Nucleic Acid and Non-Nucleic Acid Biomarker Analysis Assisted by a Spatial Confinement Effect

Nano Letters, Год журнала: 2025, Номер unknown

Опубликована: Янв. 15, 2025

CRISPR/Cas 12a system based nucleic acid and non-nucleic targets detection faces two challenges including (1) multiple crRNAs are needed for biomarkers (2) insufficient sensitivity resulted from photobleaching of fluorescent dyes the low kinetic cleavage rate a traditional single-strand (ssDNA) reporter. To address these limitations, we developed programmable DNA nanoswitch (NS)-regulated plasmonic CRISPR/Cas12a-gold nanostars (Au NSTs) reporter platform with assistance spatial confinement effect. Through simply programming target recognition sequence in NS, only one crRNA is required to detect both biomarkers. The limit decreased by ∼196-fold miRNA-375 122-fold prostate-specific antigen (PSA), respectively. Moreover, versatile evaluation PSA clinical urine samples can also be achieved, according which prostate cancer healthy groups well identified.

Язык: Английский

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Direct repeat region 3′ end modifications regulate Cas12a activity and expand its applications

Nucleic Acids Research, Год журнала: 2025, Номер 53(3)

Опубликована: Янв. 24, 2025

Abstract CRISPR-Cas12a technology has transformative potential, but as its applications grow, enhancing inherent functionalities is essential to meet diverse demands. Here, we reveal a regulatory mechanism for LbCas12a through direct repeat (DR) region 3′ end modifications and de-modifications, which can regulate LbCas12a’s cis- trans-cleavage activities. We extensively explored the effects of introducing phosphorylation, DNA, photo-cleavable linker, DNA at DR on functionality. find that temporary inhibitory function Cas12a be reactivated by modification corresponding substances, such alkaline phosphatase (ALP), immunoglobulin G (IgG), alpha-fetoprotein (AFP), exonucleases, ultraviolet radiation, glycosylases, greatly expand scope application Cas12a. Clinical demonstrated promising results in ALP, AFP, trace Epstein–Barr virus detection compared gold standard methods. Our research provides valuable insights into regulating activity significantly expands potential clinical targets, paving way future universal clustered regularly interspaced short palindromic repeats (CRISPR) diagnostic strategies.

Язык: Английский

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Engineering Multi-activator-encoded DNA Nanonet to Accelerate CRISPR-Cas12a Activation for Rapid and Sensitive Electrochemiluminescence Bioassay

Talanta, Год журнала: 2025, Номер 288, С. 127724 - 127724

Опубликована: Фев. 11, 2025

Язык: Английский

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Activator Strand Modifications in CRISPR/Cas12a: Unlocking the Potential for Casp-3-Targeted Biosensing and Imaging Analysis of Apoptosis

Analytical Chemistry, Год журнала: 2025, Номер unknown

Опубликована: Фев. 12, 2025

The CRISPR/Cas12a system has emerged as a powerful tool in biosensing due to its unique trans-cleavage activity. This study conducted an in-depth investigation of the modulatory capabilities this system, particularly focusing on 5'-end modifications activator strand, and found that introducing hairpin structure (HP) at which was designed based RESET effect, can effectively suppress strand's ability activate activity system. suppression is independent HP's relation strand type linker used (DNA, RNA or peptide). Detaching HP from restores system's These findings enrich development CRISPR/Cas12a-based biosensors, expand their application beyond DNA-based target detection peptide sequence-based recognition. Based discovery, we constructed sensitive biosensor for caspase-3 (Casp-3), key executor apoptosis, by linking with containing Casp-3 recognition site. proposed been validated sensitivity specificity detecting Casp-3, well monitoring drug-induced apoptosis through imaging living cells, providing valuable studying apoptotic process, screening drugs, assessing drug efficacy, evaluating treatment outcomes. strategy also shows promise other peptide-based targets, broadening horizons early disease biomarker timely therapeutic interventions.

Язык: Английский

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CRISPR-Cas12a/Cas13a Multiplex Bioassay for ctDNA and miRNA by Mass Spectrometry

Analytical Chemistry, Год журнала: 2025, Номер unknown

Опубликована: Фев. 20, 2025

The CRISPR-Cas system, particularly CRISPR-Cas12a and CRISPR-Cas13a, has been widely utilized in constructing various biosensors due to their "trans-cleavage" ability as a means of signal amplification. However, this universal characteristic also presents challenge for realizing multiplexed bioanalysis. Besides, potential cascading interference complicated design are notable obstacles Herein, we propose mass spectrometry method that leverages the CRISPR-Cas12a/13a system achieve simultaneous detection ctDNA miRNA. Based on properties two types nanoparticle reporter probes have engineered, using cancer-related biomarkers miR-21 our model analytes. tags, which intrinsically incorporated millions detectable atoms, combined with CRISPR-Cas12a/Cas13a system's ability, allow proposed strategy fmol-level limits without any nucleic acid amplification procedures. assay was successfully applied human serum samples, demonstrating its early disease diagnosis progression tracking.

Язык: Английский

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Dual-Accelerated Signal Amplification in Biosensing via Spatial Confining Catalytic Hairpin Assembly-Activated Spherical CRISPR/Cas12a System for Trans-Cleavage of Hairpin DNA Reporters

Analytical Chemistry, Год журнала: 2025, Номер unknown

Опубликована: Фев. 21, 2025

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression and implicated in various diseases, including cancer. Due to their critical role diagnostics, there is a growing need for sensitive, specific, rapid detection methods miRNAs. In this study, we present dual-accelerated signal amplification platform miRNA biosensing, which integrates spatial confining catalytic hairpin assembly (SC-CHA) with spherical CRISPR/Cas12a (S-CRISPR/Cas12a) system (SC-CHA@S-CRISPR/Cas12a) trans-cleavage of DNA reporters. The method employs biotinylated palindrome-rich sequence (PAS) form nanoballs, serve as scaffold the operation SC-CHA upon binding. products bind crRNA Cas 12a protein, activating S-CRISPR/Cas12a cleave reporter generate detectable fluorescence signal. uniqueness lies combined use nanoballs reporters, both significantly accelerate reaction kinetics, resulting generation. Additionally, nanostructure, integrated system, greatly enhances biostability accelerating kinetics. These features enable exhibit high sensitivity, limit (LOD) low 13.75 fM, excellent specificity, successfully distinguishing miRNA-21 from other assay also biostable, demonstrating reliable performance complex biological samples such human serum. This dual-acceleration approach offers promising solution rapid, specific potential applications early cancer diagnosis clinical monitoring.

Язык: Английский

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Recent advances in nucleic acid-functionalized metallic nanoparticles

Chemical Communications, Год журнала: 2025, Номер unknown

Опубликована: Янв. 1, 2025

Nucleic acid-functionalized metallic nanoparticles (N-MNPs) precisely integrate the advantageous characteristics of nucleic acids and nanomaterials, offering various abilities such as resistance to enzymatic degradation, penetration physiological barriers, controllable mobility, biomolecular recognition, programmable self-assembly, dynamic structure-function transformation. These properties demonstrate significant potential in field biomedical diagnostics therapeutics. In this review, we examine recent advancements construction theranostic applications N-MNPs. We briefly summarize methodologies employed conjugation with formation their superstructural assemblies. highlight representative N-MNPs diagnosis, imaging, smart delivery agents. also discuss challenges currently faced provide an outlook on future development directions application prospects.

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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Functionalization of Nucleic Acid Molecular Machines under Physiological Conditions: A Review

ACS Applied Bio Materials, Год журнала: 2025, Номер unknown

Опубликована: Апрель 1, 2025

In-situ fabrication of nucleic acid molecular machines in biological environments is desirable for smart theranostic applications. However, given the complex nature environments, integration multiple functional modules into a coordinated machine remains challenging. Recent advances nanotechnology offer solutions to these challenges. Here, we outline design principles acid–based tailored physiological conditions, drawing on recent examples. We review cutting-edge technologies that facilitate their functionalization settings, particularly presynthesis modifications using unnatural bases and postsynthesis via bioorthogonal chemistry noncovalent interactions. discuss advantages limitations suggest future directions overcome existing

Язык: Английский

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