Analytical and Bioanalytical Chemistry, Год журнала: 2024, Номер unknown
Опубликована: Ноя. 27, 2024
Язык: Английский
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
Процитировано
2
Advanced Functional Materials, Год журнала: 2025, Номер unknown
Опубликована: Янв. 26, 2025
Abstract Single‐atom nanozyme (SAZ) with peroxidase‐like activity has attracted great attention in point‐of‐care (POC) diagnostic, but the use of unstable H 2 O peroxidase catalytic reactions often leads to low detection accuracy. Thus, developing SAZ high oxidase (OD)‐like construct RNA sensing systems independent is imperative for improving Herein, a novel strategy fabricate boron‐nitrogen co‐doped zinc (ZnBNC‐SAZ) excellent OD‐like by carbonizing Zn based zeolitic boron imidazolate frameworks reported. The electron deficient B ligand can form Zn─N─B bond situ during temperature pyrolysis, which regulate electronic structure and further upshift d‐band center improve activity. With ZnBNC‐SAZ as signal generator, new method sensitive developed combining CRISPR/ cas13 system terminal deoxynucleotidyl transferase‐induced DNA extension reaction. limits RNAs are 20 aM. proposed biosensor adaptable lateral‐flow‐based readout universally applicable various programming guide RNA. Importantly, monitor cellular differentiation identify patients cervical carcinoma, showing potential application facile POC diagnosis.
Язык: Английский
Процитировано
0
Chemical Engineering Journal, Год журнала: 2025, Номер unknown, С. 160330 - 160330
Опубликована: Фев. 1, 2025
Язык: Английский
Процитировано
0
Analytical Chemistry, Год журнала: 2025, Номер unknown
Опубликована: Фев. 5, 2025
CRISPR/Cas systems have emerged as promising tools for nucleic acid detection. However, their practical applications been limited by poor specificity and the need additional preprocessing steps. Inspired concept of transformers, we found that changing forms crRNA with spatial arrangement may endow an enhanced performance Specifically, rationally designed two transformers─swap split crRNA─and they direct system cis- trans- cleavage decreased Cas binding affinity possess both DNA RNA detection abilities. Based on these findings, our strategy enabled identification clinical prostatic cancer in a one-step reaction, remarkable sensitivity 90.0% 96.0%. Our study deepens understanding introduces simple specificity, sensitivity, functionality molecular diagnosis.
Язык: Английский
Процитировано
0
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
Процитировано
0
ACS Applied Materials & Interfaces, Год журнала: 2025, Номер unknown
Опубликована: Март 28, 2025
The detection of food contamination in a swift and sensitive manner is essential for safeguarding public health. Clustered regularly interspaced short palindromic repeats (CRISPR)-based assays nucleic acid are renowned their high specificity convenient, related studies have focused on refining the Cas protein optimizing CRISPR (cr)RNAs design within CRISPR-based enhancing sensitivity detection. Our research offers innovative insights into fluorescence signal output intensity from physical standpoint, thereby presenting practical cost-effective strategy to lower thresholds assays. By layer microsphere arrays was spread onto bottom microfluidic chip enhance sample via self-assembly microspheres. Recombinase polymerase amplification (RPA) used amplify target sequences, followed by crRNA binding activate enzyme, cleaving fluorescein amidite (FAM)-labeled reporters emitting fluorescent signal. method successfully identified SARS-CoV-2 positive samples (10 clinical 8 environmental samples) distinguished them negative samples. Meanwhile, it detected 4 Shigella 5 In this study, developed exhibited limit (LoD) 75 fM (POCT with USB camera: 50 fM) 100 fM). It also demonstrated promising (100%) small-sample validation. Combined portable automated achieved using smartphone receive process signals obtained platform study not only applicable pathogens cold-chain products, but extends pathogen community hospitals resource-limited areas, providing an efficient solution rapid screening different settings. Moreover, can be changing RPA primer crRNA. This provides paradigm studying enhanced signaling holds significant potential advance commercialization use sensors.
Язык: Английский
Процитировано
0
International Journal of Molecular Sciences, Год журнала: 2024, Номер 25(23), С. 12686 - 12686
Опубликована: Ноя. 26, 2024
CRISPR-Cas system, a natural acquired immune system in prokaryotes that defends against exogenous DNA invasion because of its simple structure and easy operation, has been widely used many research fields such as synthetic biology, crop genetics breeding, precision medicine, so on. The miniature CRISPR-Cas12 an emerging genome editing tool recent years. Compared to the commonly CRISPR-Cas9 CRISPR-Cas12a, unique advantages, rich PAM sites, higher specificity, smaller volume, cytotoxicity. However, application Cas12 proteins methods improve efficiency have not systematically summarized. In this review, we introduce classification summarize structural characteristics type V cleavage mechanism five proteins. gene animals, plants, microorganisms is summarized, strategies are discussed, aiming provide reference for further understanding functional engineering modification system.
Язык: Английский
Процитировано
2
Nature Biotechnology, Год журнала: 2024, Номер unknown
Опубликована: Окт. 31, 2024
Язык: Английский
Процитировано
1
Elsevier eBooks, Год журнала: 2024, Номер unknown, С. 23 - 74
Опубликована: Ноя. 15, 2024
Язык: Английский
Процитировано
0
Analytical and Bioanalytical Chemistry, Год журнала: 2024, Номер unknown
Опубликована: Ноя. 27, 2024
Язык: Английский
Процитировано
0