INTERNATIONAL JOURNAL OF PHARMACEUTICS
·
2024
·
IF 5.3
Qian Sun,Hongqian Zhang,Feng Ding,Xue Gao,Zongwei Zhu,Chuanxu Yang
CRISPR-Cas genome editing technology holds great promise for wide-ranging biomedical applications. However, the development of efficient delivery system for CRISPR-Cas components remains challenging. Herein, we synthesized a series of ionizable lipids by conjugation of alkyl-acrylate to different amine molecules and further assembled ionizable lipid nanoparticles (iLNPs) for co-delivery of Cas9 mRNA and sgRNA. Among all the iLNP candidates, 1A14-iLNP with lipids containing spermine as amine head, demonstrated the highest cellular uptake, endosomal escape and mRNA expression in vitro. Co-delivery of Cas9 mRNA and sgRNA targeting EGFP by 1A14-iLNP achieved the highest EGFP knockout efficiency up to 70% in HeLa-EGFP cells. In addition, 1A14-iLNP displayed passive liver-targeting delivery of Cas9 mRNA in vivo with good biocompatibility. Moreover, we developed a simple method of lyophilization-mediated reverse transfection of CRISPR-Cas9 components for efficient genome editing. Therefore, the developed 1A14-iLNP and the lyophilization formulation, represent a potent solution for CRISPR-Cas9 delivery, which might broaden the future of biomedical applications of both mRNA and CRISPR-based therapies.
研究领域:
分子生物学
·
基因治疗
·
纳米医学
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DOI
PubMed
高影响因子
FOOD CHEMISTRY
·
2023
·
IF 8.8
Xuening Shi,Hang Li,Shuo Yao,Yukun Ding,Xiuzhu Lin,Hui Xu,Yi Liu,Chao Zhao,Tong Zhang,Juan Wang
The total bacterial count is an important indicator of food contamination in food safety supervision and management. Recently, the CRISPR/Cas12a system integrated with nucleic acid amplification has increasingly shown tremendous potential in microorganism detection. However, a general quantification strategy for total bacteria count based on the CRISPR/Cas12a system has not yet been developed. Herein, we established a sensitive bacterial quantification strategy based on the CRISPR/Cas12a system combined with magnetic covalent organic frameworks (MCOFs) and hybridization chain reaction (HCR). MCOFs acted as a carrier, adsorbing the ssDNA as HCR trigger sequence through π-π stacking. Then, the HCR circuit produces DNA duplexes containing the PAM sequences that activate the trans -cleavage activity of Cas12a for further signal amplification. Under the optimal conditions, the proposed method can quantify total bacteria in 50 min with a minimum detection concentration of 10 CFU/mL. The successful applications in food samples confirmed the feasibility and broad application prospects.
研究领域:
生物技术
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食品科学
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分子生物学
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产品关联
DOI
PubMed
高影响因子
BIOSENSORS & BIOELECTRONICS
·
2022
·
IF 12.545
Feng Qiu,Xiaoyan Gan,Jianglong Yao,Bingying Jiang,Ruo Yuan,Yun Xiang
Transcription factors (TFs) are key substances in regulating the transcription, replication and expression of genes, and the detection of TFs can provide valuable information to diagnose a variety of diseases. By integrating hybridization chain reaction (HCR)-activated Cas12a enzyme with bio-responsive DNA hydrogels, we propose a dual amplification and label-free homogeneous electrochemical detection method to realize sensitive nuclear factor-kappa B p50 (NF-κB p50) detection. The presence of the target molecules protects the DNA duplex probes from digesting by exonuclease III and initiates HCR to generate long double stranded DNAs that can activate the activity of RNA-guided Cas12a enzymes. The single-stranded region of the DNA linkers that crosslink the DNA hydrogels can be cleaved by the activated Cas12a to release a large number of electroactive substances embedded in the gels, which exhibit highly enhanced electrochemical signals for detecting target molecules at the detection limit of 54.1 fM. In addition, the successful interrogation of NF-κB p50 spiked into lysate of HeLa cells by such method is also verified. The established method thus shows new opportunities for sensitive and convenient monitoring of other transcription factors and biomarkers.
研究领域:
生物化学
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分子生物学
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医学诊断
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产品关联
DOI
PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
·
2022
·
IF 9.221
Zhirun Ji,Zhaoyang Shang,Muhammad Sohail,Pei Wang,Bingzhi Li,Xing Zhang,Guoxiang Chen
Protamine, an arginine-rich polycationic peptide, is a natural food preservative and the only approved antidote to heparin overdose. The detection of protamine is meaningful; however, the detection of protamine is difficult due to the lack of aromatic amino acids in its composition . Herein, we proposed a fluorometric biosensor for the sensitive detection of protamine based on the protamine-mediated regulation of CRISPR/Cas12a collateral cleavage. In the absence of protamine, the DNA probe specifically hybridizes with the Cas12a-crRNA complex, causing the activation of Cas12a. In the presence of protamine, positively charged protamine tightly binds to negatively charged DNA probes through electrostatic interaction , so that the DNA probe cannot hybridize with the Cas12a-crRNA complex. Using a short ssDNA modified with fluorophore and quencher as a reporter, the information of protamine concentration is transduced into DNA information and sensed in a signal turn-off manner. The linear range of this method in protamine detection is 0.04–3.2 μg/mL, yielding a detection limit of 0.03 μg/mL, and the entire sampling-to-response time is about 1 h. This biosensor provides a rationale for using the programmable nuclease Cas12a to detect protamine, and further advances the application of CRISPR technology in bioanalyses.
研究领域:
生物化学
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分子生物学
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医学检测
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产品关联
DOI
高影响因子
ANALYTICAL CHEMISTRY
·
2025
·
IF 6.7
Junzhuo Shan,Yixiao Sheng,Lun Luo,Wenhai Wang,Xinjiang Liu,Yi Ma,Jufang Wang
Apurinic/apyrimidinic endonuclease 1 (APE1) is a key enzyme involved in DNA repair and cellular redox regulation, and is frequently overexpressed in tumor cells. This highlights the urgent need for a rapid and high-sensitive point-of-care testing (POCT) strategy for APE1 to facilitate early cancer diagnosis. Rolling circle amplification (RCA) is a widely used isothermal DNA amplification method; however, its application in APE1 detection remains rare. Here, we introduce a versatile RCA-Lock (Rlock) conversion platform that enables the transformation of RCA-based nucleic acid detection technologies into APE1-responsive assays. Building upon this platform, we further developed a novel POCT method for APE1 detection─Rlock-mediated, CRISPR/Cas12a-driven RCA cycle (RCRE)─which, for the first time, integrates CRISPR/Cas12a with RCA into a one-pot APE1 detection system. The RCRE assay achieves a limit of detection of 8.86 × 10–4 U/mL within 30 min, while requiring minimal equipment, low cost, and no complex handling procedures. This Rlock-based conversion strategy represents a transformative advance in the field of APE1 diagnostics and offers conceptual inspiration for the design of programmable nucleic acid–based biosensors. The resulting RCRE assay significantly broadens the technological landscape for early cancer detection and paves the way for future clinical translation.
研究领域:
分子诊断学
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生物化学
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生物技术
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癌症研究
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遗传学
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分析化学
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产品关联
DOI
PubMed
高影响因子
NUCLEIC ACIDS RESEARCH
·
2023
·Vol. 51
·期 8
·4064-4077
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IF 14.9
Deng Huaping,Xu Han,Wang Yiru,Jia Ruizhen,Ma Xiaoqian,Feng Yushuo,Chen Hongmin
CRISPR (clustered regularly interspaced short palindromic repeats) technology holds tremendous promise for gene regulation and editing. However, precise control of CRISPR editing is essential to overcome its uncontrollable reaction process and excessive activity that leads to off-target editing. To overcome this problem, we engineered a photoswitch on G-quadruplex gRNA (GqRNA) for precisely controlled gene editing and expression by embedding dicationic azobenzene derivatives (AZD++). Our results demonstrated that rational design of the G-quadruplex onto crRNA conferred higher stability and sequence recognition specificity than unmodified single guide (sgRNA). Light-induced isomerization of AZD++ quickly transformed the on state of GqRNA, which facilitated rapid activation of ribonucleoprotein activity for genome editing of on-target sites in cells with excellent editing efficiency. In turn, AZD++–GqRNA promptly refolded to an off state to inhibit genomic cleavage, and limited the generation of off-target effects and by-products. Therefore, the proposed strategy of a photo-reversible modality presents a new opportunity for CRISPR-Cas9 modulation to improve its safety and applicability.
研究领域:
分子生物学
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基因工程
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生物技术
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光遗传学
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产品关联
DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2025
·
IF 6.7
Tianyi Zhang,Yingying Zhao,Cong Zhu,Xi Zhu,Xiaohua Zhu,Ye Qiu,Zhou Nie,Chunyang Lei
Viral proteases are critical molecular targets in viral pathogenesis, representing pivotal biomarkers for understanding viral infection mechanisms and developing antiviral therapeutics. This study introduces a label-free electrochemical biosensor that enables sensitive viral protease detection by integrating protease-responsive CRISPR/Cas protein switches (CasPSs) with a hemin aptamer-functionalized electrochemical interface. The biosensor’s mechanism relies on viral protease-mediated proteolysis, which leads to the release of active Cas12a proteins from CasPSs and generates amplified electrochemical responses through continuous cleavage of immobilized redox-active hemin/aptamer complexes. This biosensor achieved specific hepatitis C virus NS3/4A protease sensing with femtomolar sensitivity and could be readily expanded to other viral proteases by replacing the CasPS module. The feasibility of this biosensor was demonstrated by monitoring enterovirus 71 3C protease activities in virus-infected cell samples with different viral loads and postinfection times. This study provides a promising strategy for integrating CRISPR biosensing with electrochemical platforms, offering a helpful analytical tool for viral infection monitoring and antiviral drug screening.
研究领域:
分子生物学
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病毒学
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生物传感技术
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医学诊断
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产品关联
DOI
PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
·
2024
·
IF 8
Kai Zhang,Yuwen Bao,Changjin Ou,Guoyin Zhu,Yizhou Zhang
This study presents a novel biosensor that integrates CRISPR-Cas12a technology with DNA tile-based architectures to enhance thrombin detection. Utilizing PAM-engineered toehold switches, the biosensor dynamically activates the CRISPR-Cas12a system upon thrombin recognition, significantly improving both sensitivity and specificity. The label-free electrochemical detection method offers rapid, cost-effective analysis, making it ideal for real-time clinical applications. The biosensor achieved an impressive detection limit of 103 aM, surpassing traditional methods and setting a new standard for thrombin assays. The biosensor's robust performance in complex biological samples, such as diluted human serum, demonstrates its reliability for clinical diagnostics. This is particularly important for the early diagnosis and management of thrombotic and coagulation-related disorders. By combining advanced genetic engineering with traditional electrochemical techniques, the biosensor not only optimizes detection but also expands its potential applications across various biomedical fields. Overall, this biosensor provides a powerful and innovative tool for thrombin detection, offering enhanced diagnostic accuracy, faster patient management, and potential cost savings in healthcare. Its superior performance and adaptability highlight its promise for broader adoption in clinical diagnostics and other biosensing applications.
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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产品关联
DOI
高影响因子
BIOELECTROCHEMISTRY
·
2024
·
IF 4.8
Longjian Huang,Wenzhao Zhang,Mingxuan Liu,Yuanxun Gong,Qianli Tang,Kaihua Wang,Xianjiu Liao,Kai Zhang,Jihua Wei
We present an innovative biosensor designed for the precise identification of Escherichia coli ( E.coli ), a predominant pathogen responsible for gastrointestinal infections. E.coli is prevalent in environments characterized by substandard water quality and can lead to severe diarrhea, especially in hospital settings. The device employs entropy-driven reactions to synthesize copious amounts of double-stranded DNA (dsDNA), which, upon binding with crRNA, triggers the CRISPR/Cas12a system’s cleavage mechanism. This process results in the separation of a ferrocene (Fc)-tagged DNA strand from the electrode, enhancing the electrochemical signal for E.coli ’s rapid and accurate detection. Our tests confirm the biosensor’s ability to quantify E.coli across a dynamic range from 100 to 10 million CFU/mL, achieving a detection threshold of just over 5 CFU/mL. The development of this electrochemical biosensor highlights its exceptional selectivity, high sensitivity, and user-friendly interface for E.coli detection. It stands as a significant step forward in pathogen detection technology, promising new directions for identifying various bacterial infections through the CRISPR/Cas mechanism.
研究领域:
分子生物学
·
病原体检测
·
生物传感技术
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产品关联
DOI
PubMed
高影响因子
CHEMICAL ENGINEERING JOURNAL
·
2025
·
IF 13.2
Yuan Zhang,Kangzheng Lv,Yu Zhang,Qin Mi,Daixin Ye,Xi Chen,Juan Zhang
The emergence and robust advancement of the CRISPR/Cas system have presented novel opportunities for the development of paper-based microfluidics. In this study, the CRISPR/Cas system is firstly ingeniously integrated into a paper-based platform as an efficient signal amplification component. The Cas12a-crRNA system is integrated with nitrocellulose (NC) membrane to prepare an anchored CRISPR-Cas12a system-boosted paper-based microfluidic device, thereby increasing the local concentration of the CRISPR/Cas12a-crRNA system and enhancing catalytic efficiency. Compared with free CRISPR/Cas12a system, our explored split anchored CRISPR/Cas12a signal amplification component demonstrates a catalytic constant that is 22 times higher and a specificity constant that is 34 times greater. Further, an anchored split CRISPR/Cas12a boosted paper-based microfluidic array (ASCPMA) has been further constructed for the on-site detection of Streptococcus mutans ( S. mutans ) to assess the early risk of oral caries. ASCPMA exhibits a wide linear range from 10 CFU/mL to 10 8 CFU/mL, with a detection limit as low as 2.08 CFU/mL, and it has been successfully applied to test 140 clinical saliva samples. With rapidity, accuracy, and portability, ASCPMA will facilitate early diagnosis and prevention of dental caries.
研究领域:
生物技术
·
微流控技术
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分子诊断
·
口腔医学
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产品关联
DOI
高影响因子
VIEW
·
2024
·
IF 9.7
Jie Luo,Binpan Wang,Xiaoqi Tang,Ping Huang,Sha Yang,Shuang Zhao,Shuang Xie,Qiaofeng Li,Kai Chang,Ming Chen
Tumor-derived exosomes are crucial for early non-invasive and accurate tumor diagnosis in clinical diagnostics. The development of highly sensitive, simple, and intuitive exosome assays has sparked a research upsurge in clinical diagnostics. Here, we develop a bio-responsive intelligent DNA hydrogel loaded with CRISPR/Cas12a for universal and ultrasensitive detection of the exosomes. The aptamer serves as the target response unit and switch, competitively disintegrating the region of the DNA linkers and then Cas12a/crRNA was released and activated, resulting in a high fluorescent intensity for exosome detection at the detection limit of 119 particles/μL. Moreover, a constructed colorimetric tube is made by loading a colorimetric filter membrane on the tube lid and intelligent DNA hydrogel on the tube bottom, which enables one-pot portable colorimetric detection. Without the need for laboratory instruments and professionals, this strategy allows for naked eye detection with limit of detection as low as 10 4 particles/μL, and shows great applicability in distinguishing between healthy individuals, pretreatment patients, and post-treatment patients after obtaining a testable analyte. In this study, an ultrasensitive detection platform for exosomes that enables one-step sensing and dual signal output was introduced. The findings here suggest that this platform is a promising tool for the application of liquid biopsy based on exosomes in clinical diagnosis.
研究领域:
生物技术
·
分子生物学
·
临床诊断
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纳米技术
·
癌症研究
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产品关联
DOI
高影响因子
FOOD CHEMISTRY
·
2024
·
IF 8.5
Xiaokun Wang,Hao Ding,Yinghui Sun,Yanling Ma,Guoqing Wang,Jiadong Chen,Jaebum Choo,Lingxin Chen
To address the need for highly sensitive and reliable detection of trace ochratoxin A (OTA) in food matrices, we developed a ratiometric fluorescent aptasensor by integrating CRISPR/Cas12a, hybridization chain reaction (HCR), and horseradish peroxidase (HRP)-induced inner filter effect (IFE). The mechanism involves OTA releasing an activator that initiates CRISPR/Cas12a trans-cleavage, blocking HCR assembly. This reduces HRP levels, limiting the conversion of o -phenylenediamine (OPD) to fluorescent 2,3-diaminophenazine (DAP) (emitting at 562 nm) while maintaining strong emission from 2-amino terephthalic acid (BDC-NH 2 ) at 426 nm. The F 426 /F 562 ratio serves as a “signal-on” indicator, enabling sensitive OTA detection over 0.1 pM to 10 nM, with a detection limit of 0.0417 pM. The method exhibits excellent reproducibility, with intra-day and inter-day relative standard deviations (RSDs) of 1.91 %–3.87 % and 1.79 %, respectively, along with recovery rates of 90.1 %–110.6 % in real samples. These advantages highlight its significant potential for CRISPR/Cas-based OTA detection.
研究领域:
食品安全
·
分子生物学
·
分析化学
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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）