CELL
·
2025
·
IF 42.5
Ziqiang Zhou,Shicong Zhu,Yaqiang Hong,Guangpu Jin,Rui Ma,Fan Lin,Yiyang Zhang,Hsiang-Ying Lee,Nian Liu
Discrete genomic units can recombine into composite transposons that transcribe and transpose as single units, but their regulation and function are not fully understood. We report that composite transposons harbor bivalent histone marks, with activating and repressive marks in distinct regions. Genome-wide CRISPR-Cas9 screening, using a reporter driven by the hominid-specific composite transposon SVA (SINE [short interspersed nuclear element]-VNTR [variable number of tandem repeats]-Alu) in human cells, identified diverse genes that modify bivalent histone marks to regulate SVA transcription. SVA transcripts are critical for SVA’s cis -regulatory function in selectively contacting and activating long-range gene expression. Remarkably, a subset of bivalent SVAs is activated during erythropoiesis to boost multiple erythroid gene expression, and knocking down these SVAs leads to deficient erythropoiesis. The RNA-dependent cis -regulatory function of SVA activates genes for myelopoiesis and can contribute to aging-associated myeloid-biased hematopoiesis. These results reveal that the cis -regulatory functions of composite transposons are bivalently regulated to control cell fate transitions in development and aging.
研究领域:
分子生物学
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遗传学
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表观遗传学
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发育生物学
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细胞生物学
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血液学
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衰老研究
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JOURNAL OF HAZARDOUS MATERIALS
·
2023
·
IF 13.6
Jihua Wei,Zichun Song,Jiuying Cui,Yuanxun Gong,Qianli Tang,Kai Zhang,Xinlei Song,Xianjiu Liao
In this study, we introduce an electrochemiluminescence (ECL) sensing platform based on the "Entropy-driven triggered T7 amplification-CRISPR/Cas13a system" (EDT-Cas). This platform combines a programmable entropy-driven cycling strategy, T7 RNA polymerase, and the CRISPR/Cas13a system to amplify the determination of the SARS-CoV-2 RdRp gene. The Ti 3 C 2 T x -compliant ECL signaling molecule offers unique benefits when used with the ECL sensing platform to increase the assay sensitivity and the electrode surface modifiability. To obtain the T7 promoter, the SARS-CoV-2 RdRp gene may first initiate an entropy-driven cyclic amplification response. Then, after recognizing the T7 promoter sequence on the newly created dsDNA, T7 RNA polymerase starts transcription, resulting in the production of many single-stranded RNAs (ssRNAs), which in turn trigger the action of CRISPR/Cas13a. Finally, Cas13a/crRNA identifies the transcribed ssRNA. When it cleaves the ssRNA, many DNA reporter probes carrying -U-U- are cleaved on the electrode surface, increasing the ECL signal and allowing for the rapid and highly sensitive detection of SARS-CoV-2. With a detection limit of 7.39 aM, our method enables us to locate the SARS-CoV-2 RdRp gene in clinical samples. The detection method also demonstrates excellent repeatability and stability. The SARS-CoV-2 RdRp gene was discovered using the "Entropy-driven triggered T7 amplification-CRISPR/Cas13a system" (EDT-Cas). The developed ECL test had excellent recoveries in pharyngeal swabs and environmental samples. It is anticipated to offer an early clinical diagnosis of SARS-CoV-2 and further control the spread of the pandemic.
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ANALYST
·
2022
·Vol. 147
·期 24
·5623-5632
·
IF 5.227
Jiahai Wang,Le Luo,Yunhui Li,Lanfang Chen,Cenlin Gui,Jianji Zhu,Huizhen Li,Wenlong Wang,Daqi Chen
Solid-state nanopores have been proven as a powerful platform for label-free single-molecule analysis. However, due to its relatively low resolution and selectivity, developing biosensors with good translocation signals faces two significant challenges: (1) small-sized chemical or biological targets show difficulty in producing recognizable translocation signals because of their weak interaction with the nanopore and (2) protein interferents that widely exist in biological samples or buffers would considerably deteriorate the noise level of the nanopore, submerging the translocation signal. Herein, we demonstrate an effective way to overcome both the challenges. DNA cubes were used as signal transducers that can achieve an ultra-high (>50 : 1) signal-to-noise ratio (SNR) translocation signal, which is maintained even in protein interferent-rich buffers. A sensing strategy was constructed via hepatitis B virus (HBV) target-triggered cleavage of the component elements of the DNA cube with the assistance of the CRISPR–Cas12a technology, which caused a great drop in the translocation rate. The elements to cleave were optimized, and the sensor performance was tested in different protein stabilizer-rich buffers and human serum. Coupling with the polymerase chain reaction (PCR) pre-amplification technology, HBV-positive or -negative classification was achieved with the detection limit reaching 5 aM. It is worth noting that in our method, all reaction buffers were directly used without further optimization, which is of great help for the practical application of solid-state nanopores.
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NUCLEIC ACIDS RESEARCH
·
2021
·Vol. 49
·期 21
·12048-12068
·
IF 16.971
Jiang Zhuoran,Wang Chao,Wu Zixin,Chen Kun,Yang Wei,Deng Hexiang,Song Heng,Zhou Xiang
N6-methyladenosine (m6A) modification is the most extensively studied epigenetic modification due to its crucial role in regulating an array of biological processes. Herein, Bsu06560, formerly annotated as an adenine deaminase derived from Bacillus subtilis 168, was recognized as the first enzyme capable of metabolizing the epigenetic nucleoside N6-methyladenosine. A model of Bsu06560 was constructed, and several critical residues were putatively identified via mutational screening. Two mutants, F91L and Q150W, provided a superiorly enhanced conversion ratio of adenosine and N6-methyladenosine. The CRISPR-Cas9 system generated Bsu06560-knockout, F91L, and Q150W mutations from the B. subtilis 168 genome. Transcriptional profiling revealed a higher global gene expression level in BS-F91L and BS-Q150W strains with enhanced N6-methyladenosine deaminase activity. The differentially expressed genes were categorized using GO, COG, KEGG and verified through RT-qPCR. This study assessed the crucial roles of Bsu06560 in regulating adenosine and N6-methyladenosine metabolism, which influence a myriad of biological processes. This is the first systematic research to identify and functionally annotate an enzyme capable of metabolizing N6-methyladenosine and highlight its significant roles in regulation of bacterial metabolism. Besides, this study provides a novel method for controlling gene expression through the mutations of critical residues.
研究领域:
分子生物学
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表观遗传学
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微生物学
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基因组学
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生物化学
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Journal of Zhejiang University-SCIENCE B
·
2024
·
IF 4.7
Jia Xiaodan,Lu Ke,Liang Xufang
Neuropeptide Y receptor Y8 (NPY8R) is a fish-specific receptor with two subtypes, NPY8AR and NPY8BR. Changes in expression levels during physiological processes or in vivo regulation after ventricular injection suggest that NPY8BR plays an important role in feeding regulation; this has been found in only a few fish, at present. In order to better understand the physiological function of npy8br , especially in digestion, we used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology to generate npy8br −/− Japanese medaka ( Oryzias latipes ). We found that the deletion of npy8br in medaka larvae affected their feeding and digestion ability, ultimately affecting their growth. Specifically, npy8br deficiency in medaka larvae resulted in decreased feed intake and decreased expression levels of orexigenic genes ( npy and agrp ). npy8br −/− medaka larvae fed for 10 d (10th day of feeding) still had incompletely digested brine shrimp ( Artemia nauplii ) in the digestive tract 8 h after feeding, the messenger RNA (mRNA) expression levels of digestion-related genes ( amy, lpl, ctra , and ctrb ) were significantly decreased, and the activity of amylase, trypsin, and lipase also significantly decreased. The deletion of npy8br in medaka larvae inhibited the growth and significantly decreased the expression of growth-related genes ( gh and igf1 ). Hematoxylin and eosin (H&E) sections of intestinal tissue showed that npy8br −/− medaka larvae had damaged intestine, thinned intestinal wall, and shortened intestinal villi. So far, this is the first npy8br gene knockout model established in fish and the first demonstration that npy8br plays an important role in digestion.
研究领域:
分子生物学
·
遗传学
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水产养殖
·
生理学
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BIOSENSORS & BIOELECTRONICS
·
2026
·
IF 11.8
Lingsong Lu,Yingying Zhang,Bei Liu,Nuo zhou,Dao jun Yu,Ying Wang
Circular RNAs (circRNAs) represent an emerging family of noncoding transcripts defined by closed-loop architecture, which are now established as key participants in the etiology of tumorigenesis and malignant progression. While circRNAs show potential as therapeutic targets and biomarkers, the accurate detection of circRNAs remains challenging due to interference from homologous linear RNAs. In this study, an ultrasensitive method for detecting circ_0051240 based on the sulfhydrated toehold capture probe-initiator assembly-mediated hybridization chain reaction (HCR) and the CRISPR-Cas12a system was developed. The capture probe (with a toehold domain)-initiator strand duplex was ingeniously fabricated to identify the back-splice junction (BSJ) of circRNA. After magnetic enrichment and separation by the Fe 3 O 4 -Au@UIO-66-NH 2 nanocomposite, the initiator strand (H0) was delivered to trigger HCR. The HCR product dsDNA concatemers contained multiply repeated CRISPR-targetable DNA sites that were readily recognized by the CRISPR RNA (crRNA). This specific recognition and binding activated the CRISPR-Cas12a system's collateral endonuclease activity, leading to cleavage of the fluorophore-quencher (FQ) reporters and fluorescence emission at a characteristic wavelength. This design eliminated linear RNA-related interference and enhanced the detection of fluorescence intensity (FI). Under optimal conditions, the proposed HCR/CRISPR-Cas12a method exhibited a wide quantitative measurement range spanning from 45 pM up to 180 nM, achieving a notable limit of detection (LOD) of 0.03 pM. In this study, a novel circRNA sensing strategy capable of accurate and highly sensitive quantification of ovarian cancer-specific circRNA was reported. The proposed method exhibits acceptable performance when compared to present approaches.
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Environmental Science-Nano
·
2022
·Vol. 9
·期 9
·3417-3426
·
IF 9.473
Fan Yang,Wei Wang,Mei Zhang,Wenxin Tao,Youwang Wang,Jiameng Shi,Yuedi Ding,Minhao Xie,Sai Zhang,Zhenqiang Fan,Kai Zhang
Since the emergence of coronavirus disease 2019 (COVID-19), this highly contagious disease has ravaged the world, cumulatively causing millions of deaths and huge economic losses. As the culprit of COVID-19, severe acute respiratory syndrome β-coronavirus 2 (SARS-CoV-2) is highly infectious and pathogenic, which has caused extreme panic worldwide. Early and rapid monitoring of SARS-CoV-2 has a significant role in suppressing the pandemic and reducing the virus's lethality. In our work, we prepared a self-enhanced ruthenium complex linked to zeolitic imidazole framework-8 (ZIF-8) and used it as an electrochemiluminescence (ECL) emitter. Additionally, a double-stranded specific nuclease (DSN)-assisted target RNA cycling with catalytic hairpin assembly (CHA) signal amplification technology was used to achieve the conversion of target RNA concentration to double-stranded DNA (dsDNA) output which significantly improved the detection sensitivity of target RNA under environmental conditions and in real human serum samples. In addition, we also combined the trans-cleavage property of CRISPR–Cas12a with the adsorption property of C3N4 on a ferrocene (Fc)-labeled DNA probe and obtained target RNA-dependent ECL signals. The reliable detection protocol achieved the transformation of SARS-CoV-2 RNA concentration to ECL responses, obtaining a limit of detection (LOD) of 0.67 fM with high specificity and reproducibility, which was of guiding significance for current detection methods of mutant SARS-CoV-2 and universal RNA.
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iScience
·
2025
·
IF 4.1
Kuan Yoow Chan,Yini Yu,Yidi Kong,Ling Cheng,Renzhi Yao,Phoebe Sha Yin Chair,Ping Wang,Rong Wang,Wan-Yang Sun,Rong-Rong He,Junxia Min,Fudi Wang,Mikael Björklund
Despite wide variation, each cell type has an optimal size. Maintaining optimal size is essential for cellular fitness and function but the biological basis for this remains elusive. Here, we performed fitness analysis involving genome-wide CRISPR/Cas9 knockout data from tens of human cell lines and identified that cell size influences the essentiality of genes related to mitochondria and membrane repair. These genes also included glutathione peroxidase 4 (GPX4), which safeguards membranes from oxidative damage and prevents ferroptosis — iron-dependent death. Growth beyond normal size, with or without cell cycle arrest, increased lipid peroxidation resulting in a ferroptosis-sensitive state. Proteomic analysis revealed cell cycle -independent superscaling of endoplasmic reticulum, accumulation of iron and lipidome remodeling. Even slight increases from normal cell size sensitized proliferating cells to ferroptosis as evidenced by deep-learning-based single-cell analysis. Thus, lipid peroxidation may be a fitness trade-off which constrains cell enlargement and contributes to the establishment of an optimal cell size.
研究领域:
细胞生物学
·
基因组学
·
系统生物学
·
癌症研究
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ENZYME AND MICROBIAL TECHNOLOGY
·
2019
·
IF 3.553
Liuyan Gu,Haibo Yuan,Xueqin Lv,Guangsheng Li,Rigang Cong,Jianghua Li,Guocheng Du,Long Liu
5-Methylpyrazine-2-carboxylic acid (MPCA) is an important pharmaceutical intermediate and is used in the production of hypoglycemic agents and lipid-lowering drugs. This work aimed to develop a whole-cell biocatalytic process for the efficient synthesis of MPCA from 2, 5-dimethylpyrazine (DMP). Firstly, a whole-cell biocatalyst Escherichia coli strain was constructed by plasmid-based expression of xylene monooxygenase (XMO), benzyl alcohol dehydrogenase (BADH), and benzaldehyde dehydrogenase (BZDH) from Pseudomonas putida ATCC 33015, resulting in MPCA titer of 5.0 g/L. Then, the reaction conditions were optimized and the MPCA titer was further increased to 9.1 g/L. Next, the Ribosome Binding Site (RBS) Calculator v2.0 was used to predict and compare the translation initiation rates of the RBS sequences preceding xylM and xylA genes, encoding the two subunits of XMO. By optimizing the RBS sequences preceding xylA , the MPCA titer was increased to 10.2 g/L and the yield of MPCA on DMP reached 0.665 mol/mol. Finally, to achieve plasmid-free production of MPCA, we integrated the genes encoding for XMO, BADH and BZDH in the genome by using CRISPR/Cas9 and further fine-tuned the copy number ratios of xylM and xylA in the genome, improving the MPCA titer to 15.6 g/L and the yield of MPCA on DMP to 1.0 mol/mol. This work developed a high-yield and plasmid-free biocatalysis process for the environmentally friendly production of MPCA with 100% substrate conversion, and paved the way for the commercial production of MPCA in the future.
研究领域:
生物化工
·
合成生物学
·
代谢工程
·
基因组编辑
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Research
·
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
·
免疫学
·
组织工程
·
分子生物学
·
纳米技术
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高影响因子
MICROCHEMICAL JOURNAL
·
2026
·
IF 5.2
Lingsong Lu,Bei Liu
3D porous Fe 3 O 4 @ZIF-90 interface provides high-density DNA loading sites. • Mn 2+ -gated DNAzyme walker ensures a low-background controllable assay. • DNAzyme walker and CRISPR-Cas13a cascade enable dual signal amplification. • The biosensor demonstrated high accuracy and specificity in human serum samples. The precise quantification of Mucin-1 (MUC1) is vital for the early diagnosis and therapeutic monitoring of epithelial malignancies. In this study, a fluorescent biosensing platform based on a 3D porous metal-organic framework (MOF) and a cascaded signal amplification strategy was developed for MUC1 detection, driven by a target-induced conformational switch and Mn 2+ -dependent catalytic DNAzyme walking. Porous Fe 3 O 4 @ZIF-90 nanocomposites were used to construct the sensing interface, which co-immobilized dormant DNAzyme/aptamer duplexes and a high-density layer of substrate DNA (sub-DNA), serving as a robust platform for the subsequent walking process. Upon specific MUC1 recognition, the aptamer preferentially captures the target MUC1. The thermodynamic driving force induces the dissociation of the MUC1/aptamer complex, liberating the DNAzyme engine. Following magnetic separation to eliminate matrix interferences, the addition of Mn 2+ triggers the processive inter-molecular migration of the DNAzyme walker across the sub-DNA immobilized 3D porous Fe 3 O 4 @ZIF-90 interface via successive hybridization and site-specific cleavage of internal riboadenosine (rA) junctions. This inter-molecular walking process generates a high flux of trigger strands, which subsequently activates the CRISPR-Cas13a system for a robust trans -cleavage of dual-labeled fluorophore-quencher (FQ) reporters. The resulting degradation of FQ reporter enables sensitive MUC1 quantification through exponential fluorescence recovery. By synergizing the structure advantages of the 3D MOF with this cascaded signal amplification, the biosensor achieved a wide linear range from 0.37 nM to 500 nM with a low detection limit of 17.2 pM. Furthermore, the biosensor exhibited reliable specificity and recovery (102.28%–106.94%) in complex human serum assays. The proposed strategy effectively minimizes non-specific background leakage, demonstrating promising potential for the ultra-sensitive detection of MUC1 in complex biological matrices. Schematic presentation of the fluorescent biosensor for the determination of Mucin-1 based on a 3D porous Fe 3 O 4 @ZIF-90 interface and DNAzyme walker-mediated CRISPR-Cas13a cascade. Download: Download high-res image (117KB) Download: Download full-size image
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ANALYTICA CHIMICA ACTA
·
2025
·
IF 6
Jingzhi Zhou,Xichen Shi,Chaoqin He,Xiuwen Zheng,Ruo Yuan,Xia Yang
Background Entropy-Driven Circuits (EDC), distinguished by their spontaneous operation and absence of enzymatic reactions, represent a superior strategy for integration with CRISPR/Cas systems, as they obviate the potential for interference among various enzymes during the process of DNA amplification and CRISPR/Cas system integration. Due to the wide band gap of TiO 2 , its response to visible light is limited, and owing to its high crystallinity and exceptionally stable crystal lattice, the charge transfer (CT) process in TiO 2 is suboptimal. Results In this study, lychee-like Fe–TiO 2 was firstly prepared to serve as Raman enhanced substrate, facilitating exciton capture and separation to exhibit an excellent Surface-enhanced Raman spectroscopy (SERS) performance. It is proven that the incorporation of Fe results in a significantly narrower band gap for TiO 2 , facilitating exciton resonance. The amount of Fe in TiO 2 was optimized to fabricate a SERS biosensor for detection of miRNA-21 based on the Cas13a assisted entropy-driven system. The detection limit of miRNA was 43.88 fmol/L. Significance This work proposes a sensing strategy that integrates the CRISPR/Cas system with EDC, leveraging a semiconductor substrate exhibiting superior SERS performance to provide a stable Raman signal, thereby enabling highly sensitive detection of miRNA-21, which has a potential application in disease early warning and treatment.
研究领域:
生物传感器
·
纳米技术
·
生物化学
·
材料科学
·
分子生物学
·
分析化学
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