NUCLEIC ACIDS RESEARCH
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2022
·Vol. 50
·期 3
·1241-1255
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IF 19.16
Xiong Wei,Liu Xingyu,Qi Qianqian,Ji Huimin,Liu Fengbo,Zhong Cheng,Liu Simin,Tian Tian,Zhou Xiang
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) technology is a powerful tool in biology and medicine. However, the safety and application of this technology is hampered by excessive activity of CRISPR machinery. It is particularly important to develop methods for switching off CRISPR activity in human cells. The current study demonstrates the concept of supramolecular CRISPR-OFF switches by employing host-guest chemistry. We demonstrate that the CRISPR systems show considerable tolerance to adamantoylation on guide RNAs (gRNAs), whereas supramolecular complexation tremendously affects the function of adamantoyl gRNAs. Host–guest chemistry is demonstrated to be novel and effective tools to reduce unwanted excessive activities of CRISPR complexes in human cells. This work indicates considerable potential of supramolecular strategy for controlling and enhancing CRISPR systems.
研究领域:
遗传学
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分子生物学
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化学
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DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
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2023
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IF 6.2
Meng Cheng,Caiwei Tan,Bo Xiang,Weihong Lin,Bolin Cheng,Xuechun Peng,Yihao Yang,Yongping Lin
Visualized gene detection based on the CRISPR‐Cas12/CRISPR‐Cas13 technology and lateral flow assay device (CRISPR‐LFA) has shown great potential in point‐of‐care testing sector. Current CRISPR‐LFA methodology mainly utilizes conventional immuno-based LFA test strips, which could visualize whether the reporter probe is trans -cleaved by Cas protein, indicating the target positive detection. However, conventional CRISPR‐LFA usually produces false-positive results in target negative assay. Herein, a nucleic acid C hain H ybridization‐based L ateral F low A ssay platform, named CHLFA, has been developed to achieve the CRISPR‐CHLFA concept. Different from the conventional CRISPR‐LFA, the proposed CRISPR‐CHLFA system was established based on the nucleic acid hybridization between the GNP‐probe embedded in test strips and ssDNA (or ssRNA) reporter from CRISPR (LbaCas12a or LbuCas13a) reaction, which eliminated the requirement of immunoreaction in conventional immuno-based LFA. The assay realized the detection of 1–10 copy of target gene per reaction within 50 min. The CRISPR-CHLFA system achieved highly accurate visual detection of target negative samples, thus overcoming the false-positive problem that often produced in assays using conventional CRISPR‐LFA. The CRISPR‐CHLFA platform was further adopted for the visual detection of marker gene from SASR‐CoV-2 Omicron variant and Mycobacterium tuberculosis (MTB), respectively, and 100% accuracy for the analysis of clinical specimens (45 SASR‐CoV-2 specimens and 20 MTB specimens) was obtained. The proposed CRISPR-CHLFA system could provide an alternative platform for the development of POCT biosensors and can be widely adopted in accurate and visualized gene detection.
研究领域:
分子生物学
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生物技术
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诊断医学
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DOI
PubMed
高影响因子
CHEMICAL ENGINEERING JOURNAL
·
2025
·
IF 13.2
Yiming Zhang,Zhi Chen,Songrui Wei,Jing Wang,Yujun Zhang,Huiling Lin,Hai Fu,Yingxia Liu,Qi Gao,Han Zhang,Zhongjian Xie
Simultaneous detection of multiple microRNAs (miRNAs) is crucial for diagnosing major diseases. Conventional miRNA detection methods face limitations in multiplexing, detection cycle, sensitivity and specificity. Herein, we present a novel CRISPR-linked amplification-free miRNA photoelectrochemical (CLAMP) biosensor for the direct and simultaneous analysis of multiple miRNAs with high sensitivity and specificity. The biosensor employs multi-channel screen-printed electrodes with vertical reduced graphene oxide as the substrate for the working electrode. In particular, based on the surface plasmon resonance effect of Au nanoparticles, ZnO/Au heterojunctions with an excellent photoelectric response are constructed and modified on the surface of vertical graphene to generate photocurrent signals. The probes containing protospacer adjacent motif sites, immobilised on the Au nanoparticle surface, enable the dCas9/sgRNA complex to act as a clamp to specifically recognise and bind target sequences. This biosensor translates miRNA recognition into the effect of the dCas9/sgRNA complex on the ultrahigh photoelectric response of ZnO/Au heterojunction to “CLAMP” the target miRNAs, with no need for reverse transcription or amplification. The biosensor achieves multi-channel miRNA detection and an amol-level detection limit while demonstrating single-base recognition specificity as well as excellent stability and reproducibility during the direct detection of miRNA-10b, miRNA-155 and miRNA-21. Clinical tests revealed higher miRNA concentrations in malignant pleurisy than in non-malignant pleurisy. The CLAMP biosensor presents a substantial advancement in miRNA detection and is a promising tool for clinical diagnostics and the comprehensive analysis of multiple miRNA targets.
研究领域:
生物技术
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医学诊断
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纳米材料科学
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DOI
高影响因子
BIOSENSORS & BIOELECTRONICS
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2024
·
IF 10.7
Chengchen Tan,Guoyang Xie,Song Wu,Cailu Song,Jinhui Zhang,Xinyao Yi,Jianxiu Wang,Hailin Tang
Breast cancer is reported to be one of the most lethal cancers in women, and its multi-target detection can help improve the accuracy of diagnosis. In this work, a cluster regularly interspaced short palindromic repeats (CRISPR)-Cas13a/Cas12a-based system was established for the simultaneous fluorescence detection of breast cancer biomarkers circROBO1 and BRCA1. CRISPR-Cas13a and CRISPR-Cas12a were directly activated by their respective targets, resulting in the cleavage of short RNA and DNA reporters, respectively, thus the signals of 6-carboxyfluorescein (FAM) and 6-carboxy-xrhodamine (ROX) were restored. As the fluorescence intensities of FAM and ROX were dependent on the concentrations of circROBO1 and BRCA1, respectively, synchronous fluorescence scanning could achieve one-step detection of circROBO1 and BRCA1 with detection limits of 0.013 pM and 0.26 pM, respectively. The system was highly sensitive and specific, holding high diagnostic potential for the detection of clinical samples. Furthermore, the competing endogenous RNA mechanism between circROBO1 and BRCA1 was also explored, providing a reliable basis for the intrinsic regulatory mechanism of breast cancer.
研究领域:
分子生物学
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癌症研究
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生物技术
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医学诊断
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DOI
PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
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2022
·
IF 9.221
Ailing Su,Yuan Liu,Xiumian Cao,Weiqing Xu,Chongyang Liang,Shuping Xu
A new gene detection platform based on surface-enhanced Raman spectroscopy (SERS) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system is developed, which is applied for virus gene detections. CRISPR/Cas12a activated by CRISPR ribonucleic acid (crRNA) can recognize and lock the complementary target double-stranded deoxyribonucleic acid (dsDNA), followed by the cis -cleavage on the dsDNA and the trans -cleavage on the nearby linker single-stranded DNA (ssDNA). Target-induced trans -ssDNA cleavage leads to a failure in an aggregation behavior of the designed SERS probe pair that was bridged by the linker ssDNA, which causes the SERS intensity reduced relative to the control trial that gives much stronger SERS due to the severe aggregation state of SERS probes caused by intact linker ssDNA. This sensing method achieves the highly sensitive detections of human papillomavirus (HPV) genes in serum and pseudovirus within 40 min. The detection sensitivity of this CRISPR/Cas-SERS platform can reach pM. Owing to the flexible designs of the DNA probe and crRNA, this CRISPR/Cas-SERS sensing system is a universal gene detection tool, which can be applied for the in-vitro diagnosis field and point-of-care testing.
研究领域:
分子生物学
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医学诊断
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生物传感技术
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DOI
高影响因子
TALANTA
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2023
·
IF 6.1
Dan Zhao,Jiutang Tang,Qin Tan,Xiaohong Xie,Xin Zhao,Dingpei Xing
Constructing an ultrasensitive CRISPR/Cas-based biosensing strategy is highly significant for the detection of trace targets. Here we presented a dual-amplified biosensing method based on CRISPR/Cas13a-triggered Cas12a, namely, Cas13a-12a amplification. As proof-of-principle, the developed strategy was used for miRNA-155 detection. The target bound to the Cas13a-crRNA complex and activated the cleavage activity of Cas13a for cleaving uracil ribonucleotides (rU) in the bulge structure of blocker strand (BS), resulting in the release of primer strand (PS) from the BS modified on magnetic beads. Then, the released PS activated the cleavage activity of Cas12a to cleave single-strand DNA reporter probes, producing a significantly increased fluorescent signal. The detection limit of the Cas13a-12a amplification using synthetic miRNA-155 was as low as 0.35 fM, which was much lower than that of the only Cas13a-based assay. The applied performance of this amplification strategy was verified by accurately quantifying miRNA-155 expression levels in different cancer patients. Therefore, the developed strategy offers a supersensitive and highly specific miRNAs sensing platform for clinical application.
研究领域:
分子生物学
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医学诊断
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生物技术
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DOI
PubMed
高影响因子
TALANTA
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2025
·
IF 6.1
Yafang Shen,Xingyue Tang,Jiangqi Wang,Huang Dai,Yanna Cui,Qiuyue Hu,Yuxin Wu,Fei Jia,Guijie Hao
Recent years have witnessed the flourishing of CRISPR/Cas-based biosensors in various fields. However, most of them were developed for nucleic acid detection because non-nucleic acid targets are unable to unleash the cleavage activity of the CRISPR/Cas system directly. To circumvent this problem, activator DNA and deoxyribonuclease I (DNase I) were introduced in this research to render the CRISPR/Cas12a system as a new powerful tool for the detection of enrofloxacin (ENR), a common veterinary drug. In this biosensor, target ENR competed with DNase I- and bovine serum albumin-ENR composite-modified gold nanoparticles (DNase I-AuNPs-BSA-ENR) for the binding sites on the surface of antibody-modified magnetic nanoparticles (immuno-MNPs). Then, the captured DNase I-AuNPs-BSA-ENR degraded the activator DNA in the solution, which inhibited the activation of the CRISPR/Cas12a system. Finally, the fluorescence released by the activated CRISPR/Cas12a system was measured for the quantitative detection of ENR. The ingenious use of activator DNA and DNase I helped transduce the target recognition event into the cleavage activity of the CRISPR/Cas12a system. Moreover, the dual enzymatic amplification from DNase I and the CRISPR/Cas12a system guaranteed the sensitivity of this method with a low detection limit of 0.04 ng/mL. The developed biosensor extended the application of the CRISPR/Cas12a system for the sensitive detection of non-nucleic acid targets, providing a powerful tool in various fields such as environmental monitoring, food safety and clinical diagnosis.
研究领域:
生物技术
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食品安全
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环境监测
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临床诊断
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产品关联
DOI
PubMed
高影响因子
New Biotechnology
·
2021
·
IF 5.079
Ke Liu,Yang Gao,Zhen-Hai Li,Min Liu,Feng-Qing Wang,Dong-Zhi Wei
Efficient and convenient genetic manipulation of mycobacteria, important microorganisms in human healthcare and the pharmaceutical industry, is limited. In this study, using a model strain Mycolicibacterium neoaurum ATCC 25795, the classical bacterium for the production of valuable steroidal pharmaceuticals, a genome editing system employing CRISPR-Cas12a to achieve efficient and precise genetic manipulation has been developed. Targeted genome mutations could be easily achieved by the CRISPR-Cas12a system without exogenous donor templates, assisted by innate non-homologous end-joining (NHEJ). CRISPR-Cas12a enabled rapid one-step genomic DNA fragment deletions of 1 kb, 5 kb, 10 kb, 15 kb, 20 kb and 24 kb with efficiencies of 70 %, 30 %, 30 %, 20 %, 20 % and 10 %, respectively. Combined with the pNIL/pGOAL system, CRISPR-Cas12a successfully integrated the gene of interest into the targeted genomic site by single crossover and double crossovers with efficiencies of 100 % and 9 %, respectively, using a two-plasmid system. The robust CRISPR systems developed demonstrated strong potential for precise genome editing in M. neoaurum , including targeted deletion of DNA sequences of various lengths and integration of targeted genes into desired sites in the genome.
研究领域:
微生物学
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基因工程
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制药工业
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DOI
PubMed
高影响因子
Food Quality and Safety
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2025
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IF 4.4
Hu Jinyan,Liu Xiaogang,Liu Tiantian,Zhao Ting,Li Mingliang,Yang Qilin,Jiang Xiaofeng,Xiong Yanfei,Shen Caihong,Liu Shuangping,Mao Jian,Tu Rongkun
ObjectivesYeast plays a crucial role in fermented foods, yet the application of the CRISPR/Cas system for yeast detection has been rarely reported. The objective of this study is to develop a rapid and quantitative detection method for brewing yeast based on the CRISPR-Cas12a system, aiming to achieve higher sensitivity than the quantitative real-time polymerase chain reaction (qPCR) assay and fill the gap in the application of CRISPR for yeast detection in fermented foods.Materials and Methods: This work established one-step RPA-CRISPR/Cas12a (ORCC) detection for quantifying yeasts in fermented foods by integrating target amplification and the CRISPR system into a one-pot reaction.ResultsA numerical standardized definition was established for the quantifiable state in ORCC detection of yeasts. Using ORCC detection, which occurs at 37 °C, absolute quantification of Saccharomyces cerevisiae, Wickerhamomyces anomalus, and Zygosaccharomyces bailii can be completed in less than 45 min. Under optimal conditions, the limits of quantification and the limits of detection for three yeasts were 100 copies/μL and 1 copy/μL, with no cross-reaction observed among non-target yeasts. In pure yeast samples, ORCC detection of the target yeasts achieved a detection rate of 91.60% - 116.57%. In simulated fermentation samples, the recovery rate for spiked target yeasts using ORCC detection ranged from 92.78% to 106.90%. In original naturally fermented samples, target yeasts can be quantified by ORCC detection but not by qPCR assay. In spiked naturally fermented samples, the recovery rates of the qPCR assay were generally lower than those of ORCC detection.ConclusionORCC detection is simple, rapid, accurate, and highly sensitive for detecting three yeasts in fermented foods. It has significant potential for precisely monitoring yeast growth and fermentation process in various fermentation systems.
研究领域:
微生物学
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分子生物学
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食品科学
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DOI
高影响因子
BIOTECHNOLOGY AND BIOENGINEERING
·
2021
·Vol. 118
·期 5
·2053-2066
·
IF 4.53
Wei Lin,Tian Tian,Yongzhong Jiang,Erhu Xiong,Debin Zhu,Xiaoming Zhou
Polymerase chain reaction (PCR), a central technology for molecular diagnostics, is highly sensitive but susceptible to the risk of false positives caused by aerosol contamination, especially when an end-point detection mode is applied. Here, we proposed a solution by designing a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 eraser strategy for eliminating potential contamination amplification. The CRISPR/Cas9 engineered eraser is firstly adopted into artpcr reverse-transcription PCR (RT-PCR) system to achieve contamination-free RNA detection. Subsequently, we extended this CRISPR/Cas9 eraser to the PCR system. We engineered conventional PCR primers to enable the amplified products to contain an implanted NGG (protospacer adjacent motif, PAM) site, which is used as a code for specific CRISPR/Cas9 recognition. Pre-incubation of Cas9/sgRNA with PCR mix leads to a selective cleavage of contamination amplicons, thus only the template DNA is amplified. The developed CRISPR/Cas9 eraser, adopted by both RT-PCR and PCR systems, showed high-fidelity detection of SARS-CoV-2 and African swine fever virus with a convenient strip test.
研究领域:
分子生物学
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医学诊断
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基因编辑
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产品关联
DOI
PubMed
高影响因子
Virology Journal
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2025
·
IF 3.8
Jinlei Guo,Yunfeng Shan,Ge Hu,Xiaojuan Zhu,Kangchen Zhao,Tao Wu,Qiao Qiao,Ying Chi,Lunbiao Cui,Yiyue Ge
Background Monkeypox virus (MPXV) infection has garnered significant global attention due to its rising incidence and substantial public health implications. A rapid, sensitive, and accurate diagnostic method is urgently required to enable early intervention and effective management of MPXV outbreaks. Results In this study, we developed a novel one-step assay that integrates loop-mediated isothermal amplification (LAMP) with CRISPR/Cas12b in one-pot for the detection of MPXV. The entire detection process did not require opening the lid of the reaction tube and could be completed within 40 min using extracted viral nucleic acids, which is faster than real-time quantitative PCR (qPCR). And the results could be interpreted through either real-time fluorescence or naked-eye visualization. The limit of detection (LOD) of the assay was demonstrated to be 6.5 copies per reaction and no cross-reactivity with other pathogens such as HSV, EBV, CVA16, EV-A71, and MV was found. Furthermore, when evaluated using 113 clinical samples, the assay achieved 100% sensitivity (71/71) and 100% specificity (42/42) compared to the qPCR. Conclusions In resource-limited settings, our method requires only a portable heat block or water bath and a blue light or ultraviolet flashlight for visual detection of MPXV, making it highly accessible. The integration of LAMP and CRISPR/Cas12b provides a robust, user-friendly platform for point-of-care testing, with promising potential for the rapid molecular diagnosis of infectious diseases. Supplementary Information The online version contains supplementary material available at 10.1186/s12985-025-02780-0.
研究领域:
医学
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分子生物学
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传染病学
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DOI
PubMed
高影响因子
MICROCHIMICA ACTA
·
2024
·Vol. 191
·期 7
·1-11
·
IF 5.3
Liang Pengda,Lv Bei,Chen Ke,Li Dawei
The combination of CRISPR/Cas12a and functional DNA provides the possibility of constructing biosensors for detecting non-nucleic-acid targets. In the current study, the duplex protospacer adjacent motif (PAM) in the activator of CRISPR/Cas12a was used as a molecular switch, and a sensitive adenosine triphosphate (ATP) detection biosensor was constructed using an allosteric probe-conjugated PAM site formation in hybridization chain reaction (HCR) integrated with the CRISPR/Cas12a system (APF-CRISPR). In the absence of ATP, an aptamer-containing probe (AP) is in a stem-loop structure, which blocks the initiation of HCR. In the presence of ATP, the structure of AP is changed upon ATP binding, resulting in the release of the HCR trigger strand and the production of long duplex DNA with many PAM sites. Since the presence of a duplex PAM site is crucial for triggering the cleavage activity of CRISPR/Cas12a, the ATP-dependent formation of the PAM site in HCR products can initiate the FQ-reporter cleavage, allowing ATP quantification by measuring the fluorescent signals. By optimizing the sequence elements and detection conditions, the aptasensor demonstrated superior detection performance. The limit of detection (LOD) of the assay was estimated to be 1.16 nM, where the standard deviation of the blank was calculated based on six repeated measurements. The dynamic range of the detection was 25–750 nM, and the whole workflow of the assay was approximately 60 min. In addition, the reliability and practicability of the aptasensor were validated by comparing it with a commercially available chemiluminescence kit for ATP detection in serum. Due to its high sensitivity, specificity, and reliable performance, the APF-CRISPR holds great potential in bioanalytical studies for ATP detection. In addition, we have provided a proof-of-principle for constructing a CRISPR/Cas12a-based aptasensor, in which the PAM is utilized to regulate Cas12a cleavage activity. Graphical Abstract
研究领域:
生物化学
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分子生物学
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分析化学
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PubMed
高影响因子
危险品化学品经营许可证（带存储）