MICROCHIMICA ACTA
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2022
·Vol. 189
·期 10
·1-8
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IF 6.408
Du Yuwan,Han Dianpeng,An Zhaoxia,Wang Jiang,Gao Zhixian
Antibiotics have brought many benefits to public health systems worldwide since their first use in the last century, yet with their overuse in clinical treatment and livestock farming, new public health issues have arisen. Previously, we found in our experiments that the levels of macB genes in bovine raw milk ranked among the top of many drug resistance genes. In this paper, we present an analysis of regularly interspaced clustered short palindromic repeats (CRISPR) combined with surface-enhanced Raman scattering (SERS) technology for the detection of the drug resistance gene macB. The analysis was accomplished through the collaboration of the CRISPR system's ability to specifically identify genes and the more sensitive performance of the SERS. The analysis detects the drug resistance gene macB and does not yet require complex steps such as nucleic acid amplification. This method may prove to be an effective method for accurate detection of the drug-resistant gene macB, thus enabling more effective prevention of contamination of drug-resistant genes in food hygiene. Graphical abstract
研究领域:
微生物学
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分析化学
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食品科学
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基因工程
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PubMed
高影响因子
TALANTA
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2022
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IF 6.556
Bin Yang,Xiaowei Zeng,Jin Zhang,Jilie Kong,Xueen Fang
Delta (B.1.617.2), a highly infectious variant of SARS-CoV-2, has been sweeping the world, and threatening the safety of human life seriously. It is urgent to develop a highly selective and sensitive assay to accurately identify the SARS-CoV-2 variant Delta. In this work, we constructed a graphene/CRISPR-dCas9 electrochemical biosensor to accurately identify SARS-CoV-2 variant Delta, where the signal was further amplified by embedded electrochemical probe [Ru(phen) 2 dppz]BF 4 . This detection assay could be finished within 47 min totally, with the detection limit of 1.2 pM and good reproducibility with a C·V.% of 2.48% (n = 5). And the biosensor could selectively identify Delta among SARS-CoV-2 and other variants, including Alpha, Beta, Gamma. This assay was further validated by 26 real clinical samples, showing 100% clinical sensitivity and 100% clinical specificity, which provides a new direction for identifying other SARS-CoV-2 variants in the future.
研究领域:
生物技术
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电化学
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分子生物学
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病毒学
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PubMed
高影响因子
BIOSENSORS & BIOELECTRONICS
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2024
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IF 10.7
Han Jiang,Xiaofan Zhu,Jingran Jiao,Chao Yan,Kaiyong Liu,Wei Chen,Panzhu Qin
Accurate detection of multiple pathogens in the early stages of infection is critical for guiding treatment and saving patients' lives, but current methods are still challenged by low sensitivity, poor robustness and long turnaround times. Here, we report a CRISPR/dCas9-based hotspot self-assembling surface-enhanced Raman scattering (SERS) biosensor (called dCasSERS) and its integration with a smartphone to address these challenges. In this design, bacterial DNA was pre-amplified by loop-mediated isothermal amplification (LAMP), and the repeat sequences of the amplicons were recognized by CRISPR/dCas9, providing abundant sites for the assembly of gold nanoparticles (AuNPs) and forming numerous hotspots for SERS analysis. Using AuNPs labeled with different Raman molecules as reporters, the presence of Staphylococcus aureus ( S. aureus ), Pseudomonas aeruginosa ( P. aeruginosa ) and Escherichia coli O157:H7 ( E. coli O157:H7) could be dexterously converted into distinguishable SERS signals. CRISPR/dCas9-based amplicon-specific recognition and SERS hotspot self-assembly improved the specificity and sensitivity, enabling the biosensor to simultaneously detect three target pathogens down to 1 CFU/mL without any cross-reactivity. By introducing a rapid extraction procedure and a smartphone-integrated handheld Raman spectrometer, rapid on-site analysis of multiple pathogens could be achieved in less than 50 min. The accuracy and robustness of the biosensor were demonstrated by 500 real urine specimens. This study displays a new paradigm for CRISPR/Cas-based self-assembly of SERS hotspots and provides insight into the future development of pathogen screening tools.
研究领域:
分子生物学
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医学诊断
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纳米技术
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产品关联
DOI
PubMed
高影响因子
Food Frontiers
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2023
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IF 9.9
Yaru Li,Jiali Qiao,Zhiying Zhao,Qiang Zhang,Wenlu Zhang,Shuli Man,Shengying Ye,Kai Chen,Long Ma
It is imperative to develop practicable pathogenic bacteria detection methods. We devised a biosensor for the ultrasensitive detection of Salmonella typhimurium ( S. typhi ), termed as SCOUT-dCas9 (ultrasensitive, cross-validating, on-site, and dUal-mode test using CRISPR/dCas9). Simply, the species-specific invA gene of S. typhi was amplified using loop-mediated isothermal amplification with a biotinylated primer, which can be specifically “pulled down” by “antibody-like” dCas9-single guide RNA to form a ternary complex. SYBR Green I and streptavidin-modified alkaline phosphatase were used to functionalize them to generate fluorescent and colorimetric signals, respectively. With this strategy, the target could be dexterously converted into bimodal signals that were cross-validated to afford more reliable results. For both modes, SCOUT-dCas9 was able to detect as low as 1 CFU/mL with a dynamic range from 1 to 10 9 CFU/mL. Lastly, SCOUT-dCas9 had satisfactory selectivity and was capable of detecting S. typhi -contaminated real food samples. SCOUT-dCas9 provides a robust platform for bacterial detection.
研究领域:
生物技术
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微生物学
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食品科学
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DOI
高影响因子
Journal of Genetics and Genomics
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2024
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IF 6.6
Xiaoya Zhang,Abhisek Bhattacharya,Chunxiang Pu,Yan Dai,Jia Liu,Lang Rao,Chaoguang Tian
Histone citrullination, an important post-translational modification mediated by peptidyl arginine deiminases, is essential for many physiological processes and epigenetic regulation. However, the causal relationship between histone citrullination and specific gene regulation remains unresolved. In this study, we develop a programmable epigenetic editor by fusing the peptidyl arginine deiminase PPAD from Porphyromonas gingivalis with dCas9. With the assistance of gRNA, PPAD-dCas9 can recruit peptidyl arginine deiminases to specific genomic loci, enabling direct manipulation of the epigenetic landscape and regulation of gene expression. Our citrullination editor allows for site-specific manipulation of histone H3R2,8,17 and 26 at target human gene loci, resulting in the activation or suppression of different genes in a locus-specific manner. Moreover, the epigenetic effects of the citrullination editor are specific and sustained. This epigenetic editor offers an accurate and efficient tool for exploring gene regulation of histone citrullination.
研究领域:
表观遗传学
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分子生物学
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基因组学
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生物技术
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DOI
PubMed
高影响因子
ACS Applied Bio Materials
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2021
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IF 0
Jiahua Li,Yafeng Hao,Huizhuo Pan,Yingying Zhang,Guohui Cheng,Baona Liu,Jin Chang,Hanjie Wang
Vascular endothelial growth factor (VEGF) is the key regulator in neovascular lesions. The anti-VEGF injection is a major way to relieve retinal neovascularization and treat these diseases. However, current anti-VEGF therapeutics show significant drawbacks. The reason is the inability to effectively control its therapeutic effect. Therefore, how to controllably inhibit the VEGF target is a key point for preventing angiogenesis. Here, a CRISPR-dCas9 optogenetic nanosystem was designed for the precise regulation of pathologic neovascularization. This system is composed of a light-controlled regulatory component and transcription inhibition component. They work together to controllably and effectively inhibit the target gene’s VEGF. The opto-CRISPR nanosystem achieved precise regulation according to individual differences, whereby the expression and interaction of gene was activated by light. The following representative model laser-induced choroid neovascularization and oxygen-induced retinopathy were taken as examples to verify the effect of this nanosystem. The results showed that the opto-CRISPR nanosystem was more efficacious in the light control group (NV area effectively reduced by 41.54%) than in the dark control group without light treatment. This strategy for the CRISPR-optogenetic gene nanosystem led to the development of approaches for treating severe eye diseases. Besides, any target gene of interest can be designed by merely replacing the guide RNA sequences in this system, which provided a method for light-controlled gene transcriptional repression.
研究领域:
分子生物学
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光遗传学
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纳米技术
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眼科学
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基因治疗
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DOI
PubMed
ACS Applied Materials & Interfaces
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2025
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IF 8.2
Kaiyong Yang,Chunli Zhang,Zeyu Wang,Qiqing Huang,Jing Qian,Gaoyu Shi,Wenwen Sun,Jinqiu Wang,Yu Ji,Zhaorui Sun,Yanni Song,Xin Han
EGFR tyrosine kinase inhibitors (EGFR-TKIs) have garnered substantial clinical success in treating nonsmall cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) mutations. However, the inevitable emergence of drug resistance, frequently attributed to activation, mutation, or deletion of multiple signaling pathways, poses a significant challenge. Notably, the loss of PTEN protein expression has emerged as a pivotal mechanism fostering resistance in EGFR mutant lung cancers. Consequently, strategies aimed at upregulating PTEN expression hold great promise for restoring drug sensitivity. Leveraging the versatility, precision, and efficacy of nuclease-deactivated Cas9 (dCas9) as a transcriptional activator, we designed a CRISPR-dCas9 system to stimulate PTEN expression. To further enhance target specificity and drug delivery efficiency, we innovatively harnessed the tumor cell membrane (CCM) as a homologous targeting surface coating for our vector, thereby creating a targeted activation nanoplatform. Comprehensive in vitro and in vivo evaluations demonstrated that the synergistic interplay between gefitinib and the CRISPR-dCas9 system significantly enhanced drug sensitivity. The finding underscores the potential of our approach in addressing the issue of lung cancer resistance, offering a promising avenue for personalized and effective cancer therapies.
研究领域:
肿瘤学
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分子生物学
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基因治疗
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纳米医学
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产品关联
DOI
PubMed
高影响因子
ACS Applied Nano Materials
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2024
·
IF 5.3
Ailing Su,Yuan Liu,Weihan Sun,Chongyang Liang,Weiqing Xu,Alison Rodger,James Piper,Yuling Wang,Shuping Xu
Specifically identifying one-base mismatched gene sequences is of significance for early diagnosis, prognosis, treatment, and evaluation. However, traditional polymerase chain reaction and next-generation sequencing methods require relatively long, carefully implemented analytical procedures. Here, we report a nanoconjugate that can synchronously discriminate two types of KRAS gene mutation due to the dual-recognition mechanism of the CRISPR/dCas system with the surface-enhanced Raman scattering (SERS) response output. This method applied two sets of CRISPR/dCas9 units to collect all KRAS genes via a magnetic force and the specific mutant sequence labeled through SERS nanotags, forming a dual-clamping architecture. This sensing conjugate was sufficient to quantify the amount of the two different mutations simultaneously with a low cross-reactivity. Consequently, we deem that this sensing assay satisfies the great characteristics of a genetic analytical tool with high specificity, accuracy, and simplicity, highlighting its potential as an alternative for screening multigene events and relevant genetic biomarkers.
研究领域:
分子生物学
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纳米技术
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基因工程
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生物医学工程
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癌症研究
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DOI
高影响因子
Advanced Healthcare Materials
·
2026
·
IF 9.6
Jingjing Yang,Jiawei Yang,Kunguo Liu,Shijie Wang,Ye Hu,Juanjuan Li,Hao Wang,Dan Jiang,Xin Han
Pyroptosis triggered by pore-forming Gasdermin proteins in cancer cells facilitates anti-tumor immune activation by releasing pro-inflammatory cytokines and immunogenic contents following cellular rupture. However, selectively triggering pyroptosis in tumors still remain limited in clinical applications. Here, it is reported a microfluidic-assisted bacterial delivery system using attenuated Salmonella typhimurium VNP20009 encapsulated with metal-phenolic networks composed of ferric ions (Fe 3 + ) and tannic acid (TA) to enhance intracellular gasdermin D (GSDMD) expression through targeted CRISPR/dCas9 delivery, thereby inducing robust tumor pyroptosis. Mechanistically, this system achieves cascade amplification of pyroptotic cell death through coordinated multi-modal mechanisms. Following systemic administration, VNP20009 specifically accumulates in hypoxic tumor regions while the coated Fe 3 + –TA nanofilm undergoes pH-responsive dissolution in the acidic tumor microenvironment (TME), simultaneously generating ROS through Fenton reaction and releasing CRISPR/dCas9 system to upregulate GSDMD expression. Concurrently, the abundant flagella of VNP activate caspase-1, which in turn cleaves the overexpressed GSDMD proteins into its active form, thereby triggering robust pyroptosis in tumor cells. Taken together, by coupling bacterial adjuvanticity with ROS-mediated stress and CRISPR-driven GSDMD upregulation, this strategy achieves efficient amplification of pyroptosis and promotes antitumor immune activation.
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PubMed
高影响因子
CHEMICAL ENGINEERING JOURNAL
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2025
·
IF 13.2
Yuan Tian,Qing Xu,Ning Wang,Yanling Li,Wenyu Yang,Fei Huang,Qing Hao,Liping Zhao,Zhiruo Zhang,Fengchun Zhao,Fanglong Zhao
The intricate regulatory networks in filamentous fungi severely constrain the production of high-value natural products. Here, we developed an efficient CRISPR-dCas9-SunTag activation system to overcome transcriptional repression in Aspergillus nidulans . Through systematic optimization of GCN4 repeat numbers and implementation of P2A-mediated dCas9-SunTag/GCN4-VP64 tandem expression, this platform showed 20-fold higher activation efficiency compared to dCas9-VPR system. When deployed to regulate the emodin biosynthetic pathway, multiplexed sgRNA targeting enabled coordinated upregulation of three key biosynthetic genes mdpF , mdpG , and mdpH . This precise regulation approach minimized byproduct accumulation while boosting emodin titer to 101 mg/L, achieving 71 % higher production than the natural transcription factor overexpression. Furthermore, this optimized CRISPRa system demonstrated consistent stability during liquid fermentation in 1 L bioreactors (emodin 1.3 g/L) and solid-state fermentation (emodin 1.5 mg/g cell dry weight), highlighting its potential for scalable fungal metabolic engineering. This study establishes a modular and precise genetic toolkit for reprogramming fungal secondary metabolism, offering broad potential for the engineered biosynthesis of high-value compounds.
研究领域:
分子生物学
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基因工程
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合成生物学
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真菌生物技术
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天然产物生物合成
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DOI
高影响因子
Research
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2022
·
IF 11.036
Yanhong Wang,Fangchao Xue,Yanzhao Li,Lin Lin,Yeqin Wang,Shanlan Zhao,Xingli Zhao,Yong Liu,Ju Tan,Gang Li,Haoran Xiao,Juan Yan,Hao Tian,Min Liu,Qiao Zhang,Zhaojing Ba,Lang He,Wenyan Zhao,Chuhong Zhu,Wen Zeng
Rapid integration into the host tissue is critical for long-term patency after small diameter tissue engineering vascular grafts (sdTEVGs) transplantation. Neural recognition may be required for host integration and functionalization of the graft. However, immune rejection and inflammation hinder nerve regeneration of sdTEVGs. Here, a CRISPR/dCas9-nanocarrier was used for targeted programming of regulatory T cells (Treg cells) in situ to promote nerve regeneration of sdTEVGs by preventing excessive inflammation. Treg cells and (C-C chemokine receptor) CCR2+ macrophage recruitment occurred after transplantation. The nanodelivery system upregulated ten eleven translocation (TET2) in Treg cells in vitro. Reprogrammed Treg cells upregulated anti-inflammatory cytokines and decreased the proportion of CCR2+ macrophages. IL-6 concentrations decreased to the levels required for nerve regeneration. Implantation of CRISPR/dCas9 nanodelivery system-modified sdTEVGs in rats resulted in Treg cell editing, control of excessive inflammation, and promoted nerve regeneration. After 3 months, nerve regeneration was similar to that observed in normal blood vessels; good immune homeostasis, consistency of hemodynamics, and matrix regeneration were observed. Neural recognition promotes further integration of the graft into the host, with unobstructed blood vessels without intimal hyperplasia. Our findings provide new insights into vascular implant functionalization by the host.
研究领域:
生物医学工程
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免疫学
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组织工程
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分子生物学
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纳米技术
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DOI
PubMed
高影响因子
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