TALANTA
·
2024
·
IF 5.6
Yu Tao,Haixia Wang,Enguo Ju,Yeh-Hsing Lao,Yu Zhang,Mingqiang Li
Diseases caused by viruses, such as monkeypox virus (MPXV) and human papillomavirus (HPV), pose serious threats to human health and safety. Although numerous strategies have been constructed for detecting MPXV and HPV DNA, most methods require either laborious procedures or complicated instruments involving skilled professionals. In this research, a CRISPR-Cas12a-mediated colorimetric detection platform for MPXV and HPV sensing was constructed for the first time by applying probe DNA to reprogram the catalytic properties of molybdenum disulfide quantum dots (MoS 2 QDs). In the presence of MPXV or HPV targets, the CRISPR-Cas12a trans -cleavage activity is effectively motivated to decompose the probe DNA, leading to the suppression of enzymatic activity DNA enhancer adsorbed on MoS 2 QDs, resulting in greatly decreased catalytic behaviors. The MoS 2 QDs-DNA nanohybrids displayed prominent specificity and sensitivity, with detection limits at subpicomolar levels, as well as excellent stability and accuracy for determining MPXV and HPV DNA in human sera biosamples. Furthermore, the proposed colorimetric biosensing approach can be ensembled with a smartphone platform, allowing visible analysis of DNA targets. Taken together, this colorimetric strategy offers a novel diagnosis method for MPXV and HPV DNA detection, particularly favorable for highly endemic developing countries with restricted medical and instrumental support.
研究领域:
分子生物学
·
病毒学
·
医学诊断
✓ 已验证
产品关联
DOI
PubMed
高影响因子
CANCER RESEARCH
·
2024
·
IF 12.5
Fang Liang,Zhang Li,Wang Mengran,He Yuhao,Yang Jiao,Huang Zengjin,Tan Ying,Fang Ke,Li Jun,Sun Zhiyuan,Li Yanping,Tang Yisen,Liang Weizheng,Cui Huanhuan,Zhu Qionghua,Wu Zhe,Li Yiming,Hu Yuhui,Chen Wei
Epithelial-mesenchymal transition (EMT) is a fundamental cellular process frequently hijacked by cancer cells to promote tumor progression, especially metastasis. EMT is orchestrated by a complex molecular network acting at different layers of gene regulation. In addition to transcriptional regulation, post-transcriptional mechanisms may also play a role in EMT. Here, we performed a pooled CRISPR screen analyzing the influence of 1547 RNA binding proteins (RBPs) on cell motility in colon cancer cells and identified multiple core components of P-bodies (PBs) as negative modulators of cancer cell migration. Further experiments demonstrated that PB depletion by silencing DDX6 or EDC4 could activate hallmarks of EMT thereby enhancing cell migration in vitro as well as metastasis formation in vivo. Integrative multi-omics analysis revealed that PBs could repress the translation of the EMT-driver gene HMGA2, which contributed to PB-meditated regulation of EMT. This mechanism is conserved in other cancer types. Furthermore, endoplasmic reticulum (ER) stress was an intrinsic signal that induced PB disassembly and translational derepression of HMGA2. Taken together, this study has identified a function of PBs in regulation of EMT in cancer.
研究领域:
癌症生物学
·
分子生物学
·
细胞生物学
·
遗传学
✓ 已验证
DOI
PubMed
高影响因子
NUCLEIC ACIDS RESEARCH
·
2020
·Vol. 48
·期 20
·11773-11784
·
IF 11.502
Lin Jiao,Liu Yan,Lai Peidong,Ye Huixia,Xu Liang
A variety of nanodevices developed for nucleic acid computation provide great opportunities to construct versatile synthetic circuits for manipulation of gene expressions. In our study, by employing a two-hairpin mediated nucleic acid strand displacement as a processing joint for conditional guide RNA, we aim to build artificial connections between naturally occurring RNA expressions through programmable CRISPR/Cas9 function. This two-hairpin joint possesses a sequence-switching machinery, in which a random trigger strand can be processed to release an unconstrained sequence-independent strand and consequently activate the self-inhibitory guide RNA for conditional gene regulation. This intermediate processor was characterized by the fluorescence reporter system and applied for regulation of the CRISPR/Cas9 binding activity. Using plasmids to generate this sequence-switching machinery in situ, we achieved the autonomous genetic regulation of endogenous RNA expressions controlled by other unrelated endogenous RNAs in both E. coli and human cells. Unlike previously reported strand-displacement genetic circuits, this advanced nucleic acid nanomachine provides a novel approach that can establish regulatory connections between naturally occurring endogenous RNAs. In addition to CRISPR systems, we anticipate this two-hairpin machine can serve as a general processing joint for wide applications in the development of other RNA-based genetic circuits.
研究领域:
合成生物学
·
分子遗传学
·
基因工程
✓ 已验证
DOI
PubMed
高影响因子
Analytical Methods
·
2024
·
IF 2.7
Qiong Liu,Mei Yang,He Zhang,Wenjie Ma,Xin Fu,Huiqing Li,Sainan Gao
Tuberculosis caused by Mycobacterium bovis poses a global infectious threat to humans and animals. Therefore, there is an urgent need to develop a sensitive, precise, and easy-to-readout strategy. Here, a novel tandem combination of CRISPR/Cas12a system with dual HCR (denoted as CRISPR/Cas12a-D-HCR) was constructed for detecting Mycobacterium bovis. Based on the efficient trans-cleavage activity of the active CRISPR/Cas12a system, tandem-dsDNA with PAM sites was established using two flexible hairpins, providing multiple binding sites with CRISPR/Cas12a for further amplification. Furthermore, the activation of Cas12a initiated the second hybridization chain reaction (HCR), which integrated complete G-quadruplex sequences to assemble the hemin/G-quadruplex DNAzyme. With the addition of H2O2 and ABTS, a colorimetric signal readout strategy was achieved. Consequently, the CRISPR/Cas12a-D-HCR achieved a satisfactory detection linear range from 20 aM to 50 fM, and the limit of detection was as low as 2.75 aM with single mismatched recognition capability, demonstrating good discrimination from different bacterial species. Notably, the practical application performance via the standard addition method was verified, with the recovery ranging from 96.0% to 105.2% and the relative standard deviations (RSD) ranging from 0.95% to 6.45%. The proposed CRISPR/Cas12a-D-HCR sensing system served as a promising application for accurate detection in food safety and agricultural fields.
研究领域:
分子生物学
·
病原微生物学
·
食品安全检测
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2025
·
IF 6.7
Songkuan Zhuang,Xiaojun Huang,Muyan Diao,Ruiling Dong,Yueyu Dong,Jun Liu,Yizhen Liu
Current one-pot CRISPR diagnostics necessitate meticulous control of nucleic acid hybridization parameters or extensive modification of CRISPR components to achieve complete enzymatic silencing, a fundamental bottleneck limiting their robustness and generalizability. Here, we resolve this challenge through dynamic crRNA reconfiguration, a paradigm-shifting strategy that exploits the intrinsic structural duality of CRISPR RNA. We present CONVERT (CRISPR One-Pot Nucleic acid detection Via Engineered crRNA Reconfiguration Technology), a universal platform where nontarget intact crRNA acts as a universal suppressor, achieving complete Cas12a inhibition during RPA (recombinase polymerase amplification) by irreversible enzyme sequestration. Target detection is initiated through programmable conversion to split crRNA activators, bypassing sequence-specific design constraints entirely. This crRNA conformational switching mechanism is implemented via photocleavable linker cleavage and subsequent assembly with presupplied truncated crRNA (tcrRNA), activating trans-cleavage signal amplification. Three transformative advances emerge: First, the endogenous crRNA engineering eliminates dependency on exogenous nucleic acid blockers or chemical modifications, reducing optimization costs and time. Second, near-total enzymatic suppression ensures zero cross-talk between amplification and detection phases. Third, the unified workflow enables contamination-resistant operation through spatiotemporal control, requiring no physical partitioning. Validated for Enterocytozoon bieneusi detection, the system demonstrates attomolar sensitivity of 1 aM, 100% diagnostic concordance with qPCR, and absolute specificity against related pathogens. By effectively decoupling CRISPR regulation from sequence-context constraints through rational crRNA structural plasticity, this work establishes a generalized framework for next-generation molecular diagnostics.
研究领域:
分子生物学
·
生物工程
·
诊断技术
·
合成生物学
·
核酸扩增
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2020
·
IF 5.977
Jin Zhou,Lijuan Yin,Yanan Dong,Lei Peng,Guozhen Liu,Shuli Man,Long Ma
The severity of foodborne diseases caused by foods contaminated by pathogens or their toxins creates an urgent need for the development of specific and sensitive method for detection of bacteria. In this study, taking advantages of CRISPR-Cas13a system, namely, the crRNA programmability and Cas13a “collateral effect” of promiscuous RNase activity upon target RNA recognition, we developed a bacterial sensing strategy with the name of CCB-Detection ( C RISPR- C as13a based B acterial Detection ). Staphylococcus aureus ( S. aureus ) was chosen as a model bacteria for validating the performance of CCB-Detection. Specifically, four steps were carried out: 1) simple extraction of genome DNA ; 2) specific gene amplification by PCR; 3) in vitro transcription; and 4) the “collateral effect” cleavage of reporter RNA to report the analyte signal. It was observed that CCB-Detection was capable to successfully detect the target genomic DNA (gDNA) as low as 10 0 aM. The limit of detection (LOD) was 1 CFU/mL with a dynamic detection range of S. aureus from 10 0 to 10 7 CFU/mL. The entire sample-to-answer time for this biosensor was less than 4 h. CCB-Detection demonstrated satisfactory selectivity for S. aureus without interference from other bacteria. Furthermore, CCB-Detection was successfully applied for sensing S. aureus in real food samples with both known and unknown amounts bacteria (spiked ones and non-spiked ones) and its performance is comparable to the conventional culture-based counting method but with short assay time and high sensitivity. With desirable reliability, sensitivity, specificity and simplicity, herein proposed CCB-Detection could be extended and generalised for other bacterial detection, and has great potential to be used in a wide range of applications such as food safety inspection, disease diagnosis, environment monitoring, etc .
研究领域:
食品科学
·
微生物学
·
分子生物学
·
生物技术
✓ 已验证
DOI
PubMed
高影响因子
JOURNAL OF NANOBIOTECHNOLOGY
·
2024
·Vol. 22
·期 1
·1-15
·
IF 10.6
Wang Xiaobo,Cai Chengyuan,Lv Weiqi,Chen Kechen,Li Jiaxin,Liao Kaitong,Zhang Yanqun,Huang Hongxin,Lin Ying,Rong Zhili,Duan Xiaopin
CRISPR-based gene therapy offers precise targeting and specific editing of disease-related gene sequences, potentially yielding long-lasting treatment effects. However, efficient delivery remains a significant challenge for its widespread application. In this study, we design a novel short peptide-conjugated bioreducible polymer named TSPscp as a safe and effective delivery vector for the CRISPR system. Our results show that TSPscp markedly boosts transcriptional activation and genome editing activities of multiple CRISPR systems as confirmed by decomposition-seq and Deep-seq, which is resulted from its capability in facilitating delivery of plasmid DNA by promoting cellular uptake and lysosomal escape. Additionally, TSPscp further enhances genome editing of CRISPR by delivery of minicircle DNA, a condensed form of regular plasmid DNA. More importantly, TSPscp significantly improves delivery and genome editing of CRISPR system in vivo. In summary, our study highlights TSPscp as a promising delivery tool for CRISPR applications in vivo.Graphical Abstract
研究领域:
基因治疗
·
分子生物学
·
生物技术
✓ 已验证
产品关联
DOI
PubMed
高影响因子
TALANTA
·
2023
·
IF 6.1
Chihong Ma,Qin Zhou,Jinjin Shi,Hua Gao,Di Huang,Huimin Xue,Han Wang,Zhenzhong Zhang,Sen Yang,Junli Zhang,Kaixiang Zhang
The clustered regularly interspaced short palindromic repeats (CRISPR) system provides a new molecular diagnostic tool for construction of biosensor platforms due to its high programmability and target specificity. Herein, we developed a CRISPR-empowered electrochemical biosensor by combining the advantages of CRISPR/Cas13a and primer exchange reaction (PER), named PER-E-CRISPR, for target amplification-free and sensitive detection of miR-21. Dual-signal amplification procedures involve the binding of target miR-21 induced by CRISPR-based amplification, along with the hybridization of multiple short single-stranded DNA strands with PER concatemers. When target miR-21 is present, CRISPR/Cas13a specifically recognizes the target miRNA , triggering the trans -cleavage activity of CRISPR/Cas13a. Then Cas13a/crRNA/miRNA cleaved the predesigned ribonucleotide site in hairpin 1 (HP1) and released trigger to open hairpin 2 (HP2) modified on the electrode surface. Then PER bridge sequence contained in HP2 is exposed and hybridized with PER concatemers, following multiple short single-stranded DNA tagged with methylene blue (ssDNA-MB) bond with the PER concatemers. Under optimized conditions, PER-E-CRISPR assay for detecting miR-21 exhibits linearity in dynamic range from 10 −13 to 10 −7 M, and we obtained a limit of detection (LOD) of 30.2 fM. The established PER-E-CRISPR biosensor shows perfect practical performance in actual plasma, which may have great promising prospects for miRNA detection in the field of molecular diagnosis.
研究领域:
分子诊断
·
生物技术
·
电化学
·
纳米技术
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Nanoscale
·
2022
·Vol. 14
·期 39
·14645-14660
·
IF 8.307
Li-Min Zhai,Yan Zhao,Rui-Lin Xiao,Shi-Quan Zhang,Bao-Hua Tian,Xin-Xin Li,Rong Zhang,Ri-Sheng Ma,Hai-Xia Liang
Nuclear targeted delivery has great potential in improving the efficiency of non-viral carrier mediated genome editing. However, direct and efficient delivery of CRISPR/Cas9 plasmid into the nucleus remains a challenge. In this study, a nuclear targeted gene delivery platform based on fluorescent carbon quantum dots (CQDs) was developed. Polyethylenimine (PEI) and polyethylene glycol (PEG) synergistically passivated the surface of CQDs, providing an excitation-independent green-emitting fluorescent CQDs–PEI–PEG conjugate (CQDs–PP) with an ultra-small size and positive surface charge. Here we show that CQDs–PP could bind CRISPR/Cas9 plasmid to form a nano-complex by electrostatic attraction, which can bypass lysosomes and enter the nucleus by passive diffusion, and thereby improve the transfection efficiency. Also, CQDs–PP could deliver CRISPR/Cas9 plasmid into HeLa cells, resulting in the insertion/deletion mutation of the target EFHD1 gene. More importantly, CQDs–PP exhibited a considerably higher gene editing efficiency as well as comparable or lower cytotoxicity relative to Lipo2000 and PEI-passivated CQDs–PEI (CQDs–P). Thus, the nuclear-targeted CQDs–PP is expected to constitute an efficient CRISPR/Cas9 delivery carrier in vitro with imaging-trackable ability.
研究领域:
遗传学
·
分子生物学
·
生物医学工程
·
纳米技术
✓ 已验证
产品关联
DOI
PubMed
高影响因子
JOURNAL OF NANOBIOTECHNOLOGY
·
2025
·Vol. 23
·期 1
·1-15
·
IF 12.6
Zhang Tao,Liu Guiling,Sun Siwei,Meng Zongwu,Qiu Yuzhe,Ding Ping
The antibiotic residues pose significant risks for bacterial resistance. To address the practical requirements for rapid, accurate, and on-site detection of antibiotic residues and monitoring the abundance of associated resistance genes, we report a smartphone-integrated multi-mode platform. The platform is aimed to simultaneous, accurate, and visual quantitative detection of ampicillin (AMP) and β-lactam antibiotic resistance genes (blaTEM). Specifically, we developed a magnetically controlled fluorescence, colorimetric, and photothermal biosensor based on a magnetic separation unit (aminated modified complementary DNA chain (NH2-cDNA) loading on the surface of Ferrosoferric Oxide@polydopamine (Fe3O4@PDA, FP), FP@cDNA) and a signal unit (the aptamer nucleic acid chain modified by phosphate group linked to Prussian blue@UiO-66@manganese dioxide (PB@UiO-66@MnO2, PUM) through Zr-O-P bond, PUM@Apt), for the integrated detection of AMP and blaTEM. By utilizing complementary base pairing between FP@cDNA and PUM@Apt, along with precise aptamer recognition the AMP, we achieved the fluorescence quantitative detection of AMP by measuring the signal unit in the supernatant. Subsequently, the difference of signal units in colorimetric process leads to a varying conversion rate of oxidized 3,3’,5,5’-Tetramethylbenzidine (oxTMB), enabling the output of colorimetric and photothermal signals. The competitive binding of aptamers permitting the determination of AMP in the range of 0–160 pM with a low detection limit (0.34 pM). Additionally, in the presence of blaTEM, the activated CRISPR/Cas12a indiscriminately cleaves the single-stranded portion of the FP@DNA@PUM complex obtained by magnetic separation. A PUM-based three-signal detection scheme was established for the sensitive determination of blaTEM with the limit of detection (LOD) of 1.03 pM. The integration of smartphone-assisted analysis broadens the potential of the platform for visual detection. Notably, the innovative platform, with its excellent stability, exhibits great potential as a simple yet robust approach for the simultaneously visually monitoring antibiotics and drug resistance genes, and holds promise in the field of kit development.
研究领域:
生物化学
·
分子生物学
·
分析化学
·
医学诊断
✓ 已验证
产品关联
DOI
PubMed
高影响因子
TALANTA
·
2025
·
IF 6.1
Qiyu Zhang,Zhaoying Liu,Enze Sheng,Zhenjiang Liu,Rujin Zhao,Ming Li
To address the challenge of readily available and cost-effective monitoring, a novel aptasensor platform was proposed relying on the clustered regularly interspaced short palindromic repeats/associated protein 12a (CRISPR/Cas12a)-driven and glucosemeter-assisted system for zearalenone (ZEN). The magnetic bead (MB)-based probes of MB@Apt-cDNA and MB@invertase were prepared to provide specific recognition and efficient conversion. When ZEN presented, the released cDNA activated the cutting function of CRISPR/Cas12a for ssDNA, releasing numerous invertase to convert the environmentally friendly sucrose. Then, the generated glucose signals were positively related to ZEN levels, which quantified by a glucosemeter. The proposed aptasensor displayed an excellent sensitivity, achieving the limit of detection (LOD) and detection range of 0.218 ng/mL and 0.218−109.89 ng/mL, respectively. The platform also exhibited high specificity (with cross-reactivity values below 6.81 % for other analytes) and high accuracy for ZEN detection. The satisfactory reliability and practicability had been demonstrated through its application in real agricultural byproduct samples. The area under the curve was 0.979, with an optimal operating point being (0.125, 1.000). This study can provide an alternative pathway for improving the detection performance and accessibility to public in limited situation, suggesting the efficient CRISPR/Cas12a and green glucosemeter detection strategies for other pollutant monitoring.
研究领域:
生物传感器
·
分析化学
·
食品安全
·
分子诊断
·
环境科学
·
CRISPR技术
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2024
·
IF 6.7
Weihong Yin,Kai Hu,Yunxing Yang,Jianjian Zhuang,Zheyu Zou,Yuanjie Suo,Liping Xia,Jiale Li,Yehong Gui,Haohua Mei,Juxin Yin,Tao Zhang,Ying Mu
Escherichia coli O157:H7 (E. coli O157:H7) is a prominent pathogenic bacterium that poses serious risks to food safety and public health. Rapid and accurate detection of live E. coli O157:H7 is of great importance in food quality monitoring and clinical diagnosis. Here, we report a propidium monoazide-assisted nonamplification digital CRISPR/Cas12a assay for sensitive and rapid detection of live E. coli O157:H7. The incorporation of propidium monoazide into the method enables the selective detection of live bacteria by eliminating 98% of interference from the dead bacterial nucleic acid. Implemented on microfluidic digital chips, this method can achieve absolute quantification of nonamplified nucleic acid. The entire detection process of live bacteria can be completed within 120 min without the need for establishing a standard curve, and the sensitivity of the method reaches 1.2 × 103 CFU/mL. The method was validated using various samples, yielding results consistent with the plate counting method (Pearson’s r = 0.9490). Consequently, this method holds significant potential for applications in fields requiring live bacterial detection.
研究领域:
生物技术
·
分子生物学
·
食品科学
·
公共卫生
✓ 已验证
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）