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
·
基因工程
·
制药工业
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DOI
PubMed
高影响因子
Food Quality and Safety
·
2025
·
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
·
分子生物学
·
食品科学
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DOI
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
·
分子生物学
·
分析化学
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产品关联
DOI
PubMed
高影响因子
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
·
2024
·
IF 3.8
Zhang Runze,Wang Yuzhu,Wang Xiaoxu,Ren Honglin,Du Junzheng,Yang Yongjie,Hu Xueyu,Shi Ruoran,Zhang Bo,Li Chengwei,Lu Shiying,Li Yansong,Liu Zengshan,Hu Pan
Listeria monocytogenes ( L. monocytogenes ) is a prevalent food-borne pathogen that can cause listeriosis, which manifests as meningitis and other symptoms, potentially leading to fatal outcomes in severe cases. In this study, we developed an aptasensor utilizing carboxylated magnetic beads and Cas12a to detect L. monocytogenes . In the absence of L. monocytogenes , the aptamer maintains its spatial configuration, keeping the double-stranded DNA attached and preventing the release of a startup template and activation of Cas12a’s trans-cleavage capability. Conversely, in the presence of L. monocytogenes , the aptamer undergoes a conformational change, releasing the double-stranded DNA to serve as a startup template, thereby activating the trans-cleavage capability of Cas12a. Consequently, as the concentration of L. monocytogenes increases, the observable brightness in a blue light gel cutter intensifies, leading to a rise in fluorescence intensity difference compared to the control. This Cas12a aptasensor demonstrates excellent sensitivity towards L. monocytogenes , with a lowest detection limit (LOD) of 57.15 CFU/mL and a linear range of 4×10 2 to 4×10 7 CFU/mL ( R 2 =0.9858). Notably, the proposed Cas12a aptasensor exhibited outstanding selectivity and recovery in beef samples, and could be employed for precise monitoring. This Cas12a aptasensor not only provides a novel fluorescent and visual rapid detection method for L. monocytogenes but also offers simplicity, speed, and stability compared to previous detection methods. Furthermore, it is suitable for on-site detection of beef samples. Graphical Abstract
研究领域:
微生物学
·
分子生物学
·
食品科学
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产品关联
DOI
PubMed
高影响因子
ACS Sensors
·
2021
·
IF 7.711
Yongzhong Jiang,Menglu Hu,An-An Liu,Yi Lin,Linlin Liu,Bo Yu,Xiaoming Zhou,Dai-Wen Pang
The outbreak of COVID-19 caused a worldwide public health crisis. Large-scale population screening is an effective means to control the spread of COVID-19. Reverse transcription–polymerase chain reaction (RT-qPCR) and serology assays are the most available techniques for SARS-CoV-2 detection; however, they suffer from either less sensitivity and accuracy or low instrument accessibility for screening. To balance the sensitivity, specificity, and test availability, here, we developed enhanced colorimetry, which is termed as a magnetic pull-down-assisted colorimetric method based on the CRISPR/Cas12a system (M-CDC), for SARS-CoV-2 detection. By this method, SARS-CoV-2 RNA from synthetic sequences and cultured viruses can be detected by the naked eye based on gold nanoparticle (AuNP) probes, with a detection limit of 50 RNA copies per reaction. With CRISPR/Cas12a-assisted detection, SARS-CoV-2 can be specifically distinguished from other closely related viruses. M-CDC was further used to analyze 41 clinical samples, whose performance was 95.12%, consistent with that of an approved Clinical RT-qPCR Diagnosis kit. The developed M-CDC method is not dependent on sophisticated instruments, which makes it potentially valuable to be applied for SARS-CoV-2 screening under poor conditions.
研究领域:
分子生物学
·
病毒学
·
诊断医学
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产品关联
DOI
PubMed
高影响因子
TALANTA
·
2023
·
IF 6.1
Wenping Xing,Qian Li,Cong Han,Dongdong Sun,Zheng Zhang,Xiaona Fang,Yu Guo,Feng Ge,Wei Ding,Zhaofeng Luo,Liyun Zhang
The CRISPR/Cas systems have provided wide biosensing applications. Particularly, the aptamer-involved CRISPR/Cas sensor system powerfully expanded to non-nucleic-acid targets. However, tailoring the sequence of the aptamer to explore the relationship between affinity and the activation of CRISPR/Cas12a trans -cleavage activity has not been reported yet. Herein, we developed a series of new aptamers toward the spike protein 1(S1) of SARS-CoV-2. Surface plasmon resonance measurements showed that the affinity of these aptamers to S1 was at the nM level. Subsequently, a “SET” effect (Sequence Essential Trans -cleavage activity) is discovered for the activation of CRISPR/Cas12a trans -cleavage activity. That is, an aptamer, as the activator, sequence needs to be tailored to activate CRISPR/Cas12a efficiently. A balance should be reached between affinity and activation ability. On the one hand, high affinity ensures target recognition performance, and on the other hand, activation can achieve adequate amplification and output of recognition signals. The optimized sequence (with 27 nucleotides, for short 27-nt) not only recognizes the target with a high affinity and specificity but also can trigger the CRISPR/Cas12a trans -cleavage activity efficiently, showing an excellent detection performance in electrochemical biosensors. The detection limit for SARS-CoV-2 S1 can be low at 1.5 pg mL −1 . The new CRISPR/Cas12a-derived aptasensor also displays a remarkable ability to detect Beta, Delta, and Omicron variants but is selective toward other kinds of proteins. Above all, it is robust for point-of-care testing (POCT) in complex biological fluids, such as saliva, urine, and serum, and provides a universal and scalable detecting platform. Our results provide new insights into aptamer development and a different strategy for COVID-19 antigen detection and biosensor development.
研究领域:
分子生物学
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生物传感
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病毒学
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DOI
PubMed
高影响因子
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
·
2020
·Vol. 412
·期 3
·601-609
·
IF 3.637
Zhao Xianxian,Zhang Wenqing,Qiu Xiaopei,Mei Qiang,Luo Yang,Fu Weiling
Numerous studies have shown that exosomes are closely related to the pathogenesis of various diseases, especially cancers. Therefore, a rapid and sensitive method for exosome detection will be of great importance for the diagnosis and prognosis of diseases. We report here a method for exosome detection based on the CD63 aptamer and clustered regular interspaced short palindromic repeats (CRISPR)/Cas12a system. This method consists mainly of exosomal membrane protein recognition based on the CD63 aptamer and signal amplification based on CRISPR/Cas12a. The CD63 aptamer, as an easily adaptable nucleic acid strand, is responsible for the conversion of the amounts of exosomes into nucleic acid detection, whereas CRISPR/Cas12a is responsible for highly specific nucleic acid signal amplification. The detection range of the method was determined as 3 × 10 3 –6 × 10 7 particles per microliter. Additionally, we successfully applied this method to detect exosomes in clinical samples from both healthy individuals and patients with lung cancer, and the results were highly consistent with those obtained by nanoparticle tracking analysis. In general, this method provides a highly sensitive and specific method for the detection of exosomes and offers an avenue toward future exosome-based diagnosis of diseases.
研究领域:
生物标志物研究
·
分子诊断
·
癌症生物学
·
生物技术
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产品关联
DOI
PubMed
高影响因子
BIOELECTROCHEMISTRY
·
2025
·
IF 4.5
Qiang Tang,Jiayi Zhang,Jialuo Pang,Yingying Huang,Ying Guan,Yuanxun Gong,Qianli Tang,Kai Zhang,Xianjiu Liao
This study introduces an innovative electrochemiluminescence (ECL) biosensor for the highly sensitive and specific detection of Argonaute 2 (Ago2) activity. Ago2, a key enzyme in the RNA interference (RNAi) pathway, plays a crucial role in gene regulation, and its dysregulation is associated with diseases such as cancer and viral infections. The biosensor integrates hybridization chain reaction (HCR) amplification and the CRISPR/Cas12a system, leveraging a multi-stage signal amplification strategy. The detection mechanism begins with Ago2-mediated cleavage of a designed hairpin RNA (HP-RNA), releasing single-stranded RNA (ssRNA) that triggers HCR. This amplification step generates long DNA polymers, which serve as activators for the CRISPR/Cas12a system. Cas12a's collateral cleavage activity amplifies the signal further by cleaving a DNA reporter labeled with a ruthenium-based luminophore, enhancing the ECL output. This dual amplification strategy achieves exceptional sensitivity, with a detection limit of 0.126 aM. The biosensor demonstrates excellent specificity, distinguishing Ago2 from other Argonaute proteins, and maintains high reproducibility and stability, retaining 94 % of its signal after two weeks of storage. Real-world applicability was confirmed by accurately detecting Ago2 in spiked cell lysates, with recovery rates exceeding 100 %. The combination of HCR, CRISPR/Cas12a, and ECL establishes a robust platform for biomarker detection, offering superior sensitivity and adaptability for clinical diagnostics, disease monitoring, and therapeutic evaluation. This biosensor represents a significant advancement in the development of next-generation diagnostic tools.
研究领域:
生物传感技术
·
分子生物学
·
临床诊断
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产品关联
DOI
PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
·
2020
·
IF 7.1
Lei Peng,Jin Zhou,Guozhen Liu,Lijuan Yin,Siyu Ren,Shuli Man,Long Ma
CRISPR-Cas based detection platform opens up a new avenue for biosensing owing to its programmability , sequence specificity and high base resolution upon nucleic acid targets. In current study, we utilised the target DNA-induced non-specific single-stranded DNA cutting activity ( trans -cleavage) of CRISPR-Cas12a to fabricate an aptamer mediated fluorescent biosensor for sensitive and selective detection of adenosine triphosphate (ATP). In this sensor, we designed an ATP-binding aptamer as the target ssDNA for Cas12a, thereby in the presence of ATP, the ATP-binding aptamer would be occupied by ATP, and less target ssDNA can be processed by Cas12a, which gave rise to an ATP concentration dependent change in fluorescence resulted from trans -cleavage of doubly labeled ssDNA reporter. In this way, we successfully “translated” ATP signals to nucleic acid signals that can be amplified by Cas12a process. The dynamic detection range was from 1 μM to 200 μM and the limit of detection was 400 nM. The entire sample-to-answer time for this biosensor was around 40 min. Overall, this novel biosensor balanced sensitivity, specificity, detection time and ease of use. Our proposed biosensor provided a principle-of-proof for detecting small molecule by using CRISPR-Cas12a system, which would unlock its potential and further its futuristic applications in the field of diagnostics.
研究领域:
分子生物学
·
生物传感技术
·
诊断学
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DOI
高影响因子
Analytical Methods
·
2023
·Vol. 15
·期 17
·2105-2113
·
IF 3.1
Furong Zhao,Pei Wang,Haoxuan Wang,Sirui Liu,Muhammad Sohail,Xing Zhang,Bingzhi Li,He Huang
The spread of the monkeypox virus (MPXV) from Central and West Africa to previously non-endemic regions has caused a global panic. In this context, the rapid, specific, and ultrasensitive detection of MPXV is crucial to contain its spread, though such technology has seldom been reported. Herein, we proposed an MPXV assay combining recombinase-aided amplification (RAA) and CRISPR/Cas12a. This assay targeted the highly conserved MPXV F3L gene and demonstrates a low detection limit (LOD) of 101 copies per μL. By leveraging the high specificity nature of RAA and CRISPR/Cas12a, we rationally optimized probes and conditions to achieve high selectivity that differentiates MPXV from other orthopox viruses and current high-profile viruses. To facilitate on-site screening of potential MPXV carriers, a kit integrating lateral flow strips was developed, enabling naked-eye MPXV detection with a LOD of 104 copies per μL. Our RAA-Cas12a-MPXV assay was able to detect MPXV without the need for sophisticated operation and expensive equipment. We believe that this assay can be rapidly deployed in emerging viral outbreaks for on-site surveillance of MPXV.
研究领域:
病毒学
·
分子诊断
·
生物技术
✓ 已验证
DOI
PubMed
高影响因子
ACS Applied Materials & Interfaces
·
2023
·
IF 9.5
Shaoqiong Feng,Hanjun Chen,Ziao Hu,Tingting Wu,Zhihong Liu
MicroRNA (miRNA) is a promising biomarker for the diagnosis, monitoring, and prognostic evaluation of diseases, especially cancer. The existing miRNA detection methods usually need external instruments for quantitative signal output, limiting their practical applications in point-of-care (POC) settings. Here, we propose a distance-based biosensor through a responsive hydrogel, in combination with a CRISPR/Cas12a system and target-triggered strand displacement amplification (SDA) reaction for visual quantitative and sensitive measurement of miRNA. The target miRNA is first converted into plenty of double-stranded DNA (dsDNA) via target-triggered SDA reaction. Then, the dsDNA products trigger the collateral cleavage activity of CRISPR/Cas12a, leading to the release of trypsin from magnetic beads (MBs). The released trypsin can hydrolyze gelatin, and hence the permeability of gelatin-treated filter paper is increased, resulting in a visible distance signal on a cotton thread. Using this system, the concentration of the target miRNA can be quantified visually without any assistance of instruments, and a detection limit of 6.28 pM is obtained. In addition, the target miRNA in human serum samples and cell lysates can also be detected accurately. Owing to the characteristics of simplicity, sensitivity, specificity, and portability, the proposed biosensor provides a new tool for miRNA detection and holds great promise in POC applications.
研究领域:
生物技术
·
分子诊断
·
医学检验
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DOI
PubMed
高影响因子
Biomolecules
·
2025
·Vol. 15
·期 3
·315
·
IF 4.8
Dongxia Pan,Mukaddas Mijit,Hui Wang,Chaoqun Sun,Bantan Pingcuo,Zhixue Yu,Benhai Xiong,Xiangfang Tang
The A-to-G mutation (FecB) in theBMPR1Bgene is strongly linked to fertility in sheep, significantly increasing ovulation rates and litter sizes compared to wild-type populations. The rapid and reliable screening of theFecBgene is therefore critical for advancing sheep breeding programs. This study aimed to develop a fast and accurate method for detecting theFecBmutation and genotyping the gene to enhance sheep reproduction and productivity. To achieve this, we integrated the CRISPR-Cas12a system with an optimized amplification refractory mutation system (ARMS). A similar DNA origami technique-based fluorescence reporter nanotree structure was synthesized using gold nanomagnetic beads as carriers to amplify the fluorescence signal further. The resulting biosensing platform, termed CRISPR-ARMS, demonstrated excellent sensitivity for detectingFecBmutations, with a detection limit as low as 0.02 pmol. Therefore, this innovative approach shows great promise for single-base mutation detection and represents a pioneering tool for high-yield genetic screening.
研究领域:
分子生物学
·
遗传学
·
动物科学
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产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）