ANALYTICA CHIMICA ACTA
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2023
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IF 6.2
Jiuying Cui,Qisheng Luo,Cheng Wei,Xiandong Deng,Hongqu Liang,Jihua Wei,Yuanxun Gong,Qianli Tang,Kai Zhang,Xianjiu Liao
Background Escherichia coli ( E.coli ) is both a commensal and a foodborne pathogenic bacterium in the human gastrointestinal tract , posing significant potential risks to human health and food safety. However, one of the major challenges in E.coli detection lies in the preparation and storage of antibodies. In traditional detection methods, antibodies are indispensable, but their instability often leads to experimental complexity and increased false positives. This underscores the need for new technologies and novel sensors. Therefore, the development of a simple and sensitive method for analyzing E.coli would make significant contributions to human health and food safety. Results We constructed an electrochemical biosensor based on triple-helical DNA and entropy-driven amplification reaction (EDC) to inhibit the cleavage activity of Cas12a, enabling high-specificity detection of E.coli . Replacing antibodies with nucleic acid aptamers (Apt) as recognition elements, we utilized the triple-helical DNA generated by the binding of DNA2 and DNA5/DNA6 double-helical DNA through the entropy-driven amplification reaction to inhibit the collateral cleavage activity of clustered regularly interspaced short palindromic repeats gene editing system (CRISPR) and its associated proteins (Cas). By converting E.coli into electrical signals and recording signal changes in the form of square wave voltammetry (SWV), rapid detection of E.coli was achieved. Optimization of experimental conditions and data detection under the optimal conditions provided high sensitivity, low detection limits, and high specificity. Significance With a minimal detection limit of 5.02 CFU/mL and a linear range of 1 × 10 2 - 1 × 10 7 CFU/mL, the suggested approach was successfully verified to analyze E.coli at various concentrations. Additionally, after examining E.coli samples from pure water and pure milk, the recoveries ranged between 95.76 and 101.20%, demonstrating the method's applicability. Additionally, it provides a feasible research direction for the detection of pathogenic bacteria causing other diseases using the CRISPR/Cas gene editing system.
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高影响因子
MICROCHEMICAL JOURNAL
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2023
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IF 4.8
Xianfeng Wang,Chengxiang Chu,Yuanyi Deng,Yi Ma,Mei Yang,Huibo Luo,Danqun Huo,Changjun Hou
Accurate identification of Staphylococcus aureus ( S. aureus ) is crucial for food safety and clinical diagnosis. Particularly, identifying drug-resistant strains guides effective treatment and prevention of infectious diseases. Herein, we presented a highly efficient programmable CRISPR/Cas12a system for identification of methicillin-resistant S. aureus (MRSA). Genus-specific gene (spec gene) and methicillin-resistant gene (mecA gene) of S. aureus were amplified by PCR. According to the sequences of spec gene and mecA gene, corresponding programmable crRNAs were designed and prepared to construct CRISPR/Cas12a system, which recognized and bound to the PCR products through complementary interactions to activate the trans -cleavage activity of Cas12a. This led to the indiscriminate digestion of surrounding FQ-reporter, releasing a distinct fluorescence signal for MRSA identification. In addition, we devised a pair of AuNPs-DNA probes for colorimetric recognition of S. aureus . Target-triggered trans -cleavage for linker probe led to an non-aggregation of AuNPs-DNA probes pair. This led to a noticeable appearance of red color, enabling naked-eye detection. Thus, MRSA identification was realized with remarkable sensitivity and exceptional selectivity. Successful identification of MRSA in clinical samples validated the practical applications of the proposed CRISPR/Cas12a assay, establishing it as a universal platform for diagnosing pathogenic microorganisms with versatility and compatibility.
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ANALYTICAL CHEMISTRY
·
2023
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IF 7.4
Yin Zhao,Dongjuan Chen,Zhichen Xu,Tao Li,Jiang Zhu,Rui Hu,Guoyong Xu,Ying Li,Yunhuang Yang,Maili Liu
Fast, simplified, and multiplexed detection of human papillomaviruses (HPVs) is of great importance for both clinical management and population screening. However, current HPV detection methods often require sophisticated instruments and laborious procedures to detect multiple targets. In this work, we developed a simple microfluidic dual-droplet device (M-D3) for the simultaneous detection of HPV16 and HPV18 by combining the CRISPR-Cas12a system and multiplexed recombinase polymerase amplification (RPA) assay. A new approach of combining pressure/vacuum was proposed for efficient droplet generation with minimal sample consumption. Two groups of droplets that separately encapsulate the relevant Cas12a/crRNA and the fluorescent green or red reporters are parallelly generated, followed by automatic imaging to discriminate the HPV subtypes based on the specific fluorescence of the droplets. The M-D3 platform performs with high sensitivity (∼0.02 nM for unamplified plasmids) and specificity in detecting HPV16 and HPV18 DNA. By combining the RPA and Cas12a assay, M-D3 allows on-chip detection of HPV16 and HPV18 DNA simultaneously within 30 min, reaching a detection limit of 10–18 M (∼1 copy/reaction). Moreover, the outstanding performance of M-D3 was validated in testing 20 clinical patient samples with HPV infection risk, showing a sensitivity of 92.3% and a specificity of 100%. By integrating the dual-droplet generator, CRISPR-Cas12a, and multiplexed RPA, the M-D3 platform provides an efficient way to discriminate the two most harmful HPV subtypes and holds great potential in the applications of multiplexed nucleic acid testing.
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STAR Protocols
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2022
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IF 0
Meixia Wang,Fang Yan,Huanbin Zhou
Summary Base editing is a precision genome-editing approach that is widely utilized to generate single-nucleotide variants (SNVs) in genomes. Here, we present a protocol to perform targeted adenine (A)-to-guanine (G) substitution in rice using adenine base editor (ABE). We detail the design of sgRNA, CRISPR plasmid construction, rapid genetic transformation of rice, and genotyping of editing events. This protocol can be applied to cytosine base editing in rice as well. For complete details on the use and execution of this protocol, please refer to Yan et al. (2021). 1
研究领域:
植物生物学
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基因组编辑
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分子遗传学
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ACS Sensors
·
2022
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IF 9.618
Haiyang Yu,Huibin Zhang,Jinhua Li,Zheng Zhao,Minhua Deng,Zhanpeng Ren,Ziqin Li,Chenglong Xue,Mitch Guijun Li,Zhaowei Chen
The disease caused by severe acute respiratory syndrome coronavirus, SARS-CoV-2, is termed COVID-19. Even though COVID-19 has been out for more than two years, it is still causing a global pandemic. Due to the limitations of sample collection, transportation, and kit performance, the traditional reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method has a long detection period and high testing costs. An increased risk of infection is inevitable, since many patients may not be diagnosed in time. The CRISPR-Cas13a system can be designed for RNA identification and knockdown, as a promising platform for nucleic acid detection. Here, we designed a solution-gated graphene transistor (SGGT) biosensor based on the CRISPR-Cas13a system. Using the gene-targeting capacity of CRISPR-Cas13a and gate functionalization via multilayer modification, SARS-CoV-2 nucleic acid sequences can be quickly and precisely identified without the need for amplification or fluorescence tagging. The limit of detection (LOD) in both buffer and serum reached the aM level, and the reaction time was about 10 min. The results of the detection of COVID-19 clinical samples from throat swabs agree with RT-PCR. In addition, the interchangeable gates significantly minimize the cost and time of device fabrication. In a nutshell, our biosensor technology is broadly applicable and will be suitable for point-of-care (POC) testing.
研究领域:
生物技术
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纳米技术
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分子生物学
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医学诊断
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高影响因子
BIOMATERIALS
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2020
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IF 10.317
Chengli Yang,Yuyin Fu,Cheng Huang,Danrong Hu,Kai Zhou,Ying Hao,Bingyang Chu,Yun Yang,Zhiyong Qian
Photodynamic therapy (PDT) is a relatively safe and clinically promising treatment to combat primary tumors, especially epidermal carcinoma, while has negligible effects on distant metastasis. Therefore, this work reports a multifunctional nanosystem ( [email protected] ) exerting a combined photodynamic and immunotherapy to amplify the therapeutic effect on primary tumors and distant metastasis. Specifically, this nanosystem was obtained by electrostatic adsorption of a negatively charged hyaluronic acid “shell” with a positively charged “core” consisting of the CRISPR-Cas9 system targeting the Ptpn2 gene (Cas9-Ptpn2) and a modified mitochondria-targeting chlorin e6 (TPP-PEI-Ce6). Cell experiments demonstrated that the [email protected] nanoparticles possessed very high transfection efficiency on B16F10 cells, and TPP-PEI-Ce6 in the nanoparticles resulted in a significant PDT efficacy due to the efficient singlet oxygen generation in mitochondria under laser-irradiation. The accumulation of the nanoparticles in the tumor by active and passive tumor-targeting in vivo led to the disruption of the Ptpn2 gene by the Cas9-Ptpn2 plasmids in the nanocarriers, thus sensitizing tumors to immunotherapy by the increase of the IFN-γ and TNF-α signaling and the promotion of the proliferation of CD8 + T cells . In addition, Hyaluronidase was administered in advance to destroy the hyaluronic acid in the condensed extracellular matrix and to remove the hyaluronic acid “shell” from the nanosystem, subsequently leading to an enhanced penetration of oxygen and therapeutic agents. Fortunately, the primary and distant tumors in the experimental animals were remarkably inhibited after the combination of PDT-immunotherapy, thus, this easy-to-built nanomedicine could be used as a potential combination therapy against tumors.
研究领域:
癌症治疗
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纳米技术
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分子生物学
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免疫学
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ACS Applied Materials & Interfaces
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2026
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IF 7.8
Yuduo Gao,Yiqing Chen,Zhenghao Hu,Ziyue Wang,Dongchang Sun,Chengfan Wu,Qi Shuai,Yunfeng Yan
Heat shock protein 47 (HSP47) regulates collagen synthesis and fibrosis, which makes it a promising target for controlling pulmonary fibrosis. However, challenges such as cell-selective and efficient drug delivery continue to impede the verification and translation of HSP47-targeted therapies. The CRISPR/Cas9 system can effectively inhibit the overexpression of HSP47 within fibrotic lesions while significantly reducing off-target effects. This study develops a class of lipid nanoparticles (LNPs) capable of codelivering Cas9 mRNA and sgHSP47 to fibroblasts. By incorporation of an additional cationic effector molecule, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), these LNPs achieve differential delivery across various cell lines and markedly enhance uptake efficiency in fibroblasts. This system mediates effective HSP47 knockdown in L929 cells and the lungs of C57BL/6 mice, leading to reduced collagen deposition and fibroblast activation in bleomycin-induced idiopathic pulmonary fibrosis (IPF) mice. This LNP platform holds substantial potential for gene editing-based IPF therapy and provides valuable insights for the efficient and selective delivery of CRISPR/Cas9 systems.
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BIOELECTROCHEMISTRY
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2026
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IF 4.9
Mingzhu Yang,Dujuan Li,Ao Wang,Yifan Zhang,Jiale Ouyang,Xinyi Wang,Liang Hong,Dingyi Tong,Minghua Wang,Jianhan Lin,Qiong Luo,Gaofeng Wang
Breast cancer, a globally prevalent malignancy in women, requires early diagnosis to improve patient outcomes, where ctDNA serves as a key biomarker. In the present study, an electrochemical biosensor based on CRISPR/Cas12a and Au@UiO-66 nanozyme synergistic dual amplification was developed to achieve ultrasensitive detection of breast cancer marker ctDNA. Au@UiO-66-modified ssDNA probes were anchored to the gold electrode via thiol groups. The intact ssDNA probe anchors Au@UiO-66 nanozyme to the electrode, where its peroxidase-like activity efficiently catalyzes H 2 O 2 reduction to generate amplified reduction peak currents. Target ctDNA activates Cas12a's trans-cleavage capacity by binding specifically to crRNA. Under the presence of the target, Au@UiO-66 nanozymes are released, resulting in a discernible drop in the peak current linked to H 2 O 2 reduction. Benefiting from the optimized experimental conditions, the biosensor demonstrates a broad linear detection range (from 10 fM to 10 nM) for the target ctDNA, along with an exceptionally low detection limit of 6.14 fM. Successful detection in human serum samples demonstrates its practicality. The platform's high specificity is attributed to the programmable crRNA design, enabling detection of specified DNA sequences and showcasing significant adaptability for diagnosing multiple genetic targets, highlighting its potential in clinical cancer diagnosis.
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BIOSENSORS & BIOELECTRONICS
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2026
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IF 11.8
Jihua Wei,Jiahui Wang,Runze Wu,Jiayi Zhang,Haozhen Ren,Qianli Tang,Longjian Huang,Kai Zhang,Xianjiu Liao
Sensitive and reliable detection of DNA repair enzymes is critical for early cancer diagnosis and therapeutic monitoring. Herein, we report a novel electrochemiluminescence (ECL) biosensor for ultrasensitive detection of Flap Endonuclease 1 (FEN1), integrating an aptakiss-assisted CRISPR/Cas13a signal amplification strategy with a perovskite-based nanocomposite sensing interface. Specifically, a hybrid nanomaterial composed of CsPbBr 3 nanocrystals coated with polydopamine (PDA) and decorated with gold nanoparticles (AuNPs) was constructed to form a core–shell–satellite structure (CsPbBr 3 @PDA@AuNPs). The PDA coating enhanced the aqueous stability of CsPbBr 3 and introduced functional groups for probe attachment, while the AuNPs facilitated electron transfer and signal amplification. Upon recognition and cleavage of a flap-structured DNA substrate by FEN1, a downstream transcription reaction was initiated to generate RNA triggers, which in turn activated the Cas13a system. Cas13a cleaved a surface-tethered RNA strand required for forming the aptakiss complex, thereby removing ferrocene-based ECL quenching and restoring strong luminescence. The proposed biosensor exhibited an excellent detection limit of 1.73 fM, with high selectivity against non-specific nucleases, and demonstrated remarkable reproducibility and long-term stability. This study not only presents a powerful biosensing platform for FEN1 but also highlights the potential of CsPbBr 3 @PDA@AuNPs as a versatile ECL-active material. The modular design and tunable functionality of the nanocomposite enable broad applicability in detecting various biomolecules, paving the way for the development of advanced perovskite-based ECL biosensors for clinical and environmental diagnostics.
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高影响因子
MICROCHEMICAL JOURNAL
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2026
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IF 5.2
Yaqi Wang,Xinning Wang,Xin Ji,Yunxuan Weng,Xiaoying Zhao
Zearalenone (ZEN) is a mycotoxin that poses severe health risks to humans and livestock through contaminated cereals and feed. In this study, a hydrogel-based sensor was developed for sensitive visual detection of ZEN. The sensor integrates a DNA aptamer-crosslinked polyacrylamide hydrogel encapsulating gold nanoparticles (AuNPs) with a rolling circle amplification (RCA)-assisted CRISPR/Cas12a system for signal enhancement. Upon ZEN binding, the released complementary DNA triggers RCA, generating repetitive DNA strands that activate Cas12a. The activated Cas12a efficiently cleaves numerous single-stranded DNA crosslinkers within the hydrogel, leading to substantial dissociation and release of AuNPs for colorimetric readout. This strategy achieves high specificity and a significantly improved detection limit of 1.5 nM for ZEN, which is over 3000-fold lower than that of the aptamer-hydrogel system without amplification. The sensor enables rapid, simple, and cost-effective on-site quantification of ZEN, with the detection sensitivity and linear range well suited for food safety monitoring applications.
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Materials Today Bio
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2025
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IF 10.2
Yichen Liu,Dahai Hou,Hui Wang,Qiuying Yan,Yunzhu Xu,Qibiao Wu,Dan Dong,Weixing Shen,Weiwei Tao,Haibo Cheng,Dongdong Sun
Colorectal cancer, a highly invasive malignancy, poses significant treatment challenges due to the immunosuppressive tumor microenvironment. Tumor-associated macrophages, which primarily exhibit the M2 phenotype, play a key role in cancer progression and immune evasion. Reprogramming these M2 macrophages to the tumor-suppressive M1 phenotype represents a promising therapeutic approach to enhance antitumor immunity. In this study, we developed a calcium lactate nanoparticle-based delivery system (CLBRP) to codeliver bufalin, a potent Na + /K + -ATPase inhibitor with anticancer properties, and CRISPR-Cas9, which targets the immune checkpoint CD47. The system releases its payload under low pH conditions typical of the tumor microenvironment, increasing extracellular Ca 2+ and intracellular osmotic pressure, thereby inducing pyroptosis and immunogenic cell death. Bufalin induces cancer cell apoptosis, inhibits Na + /K + -ATPase, and triggers pyroptosis, which activates a strong immune response. Additionally, bufalin and D-lactic acid synergistically promote macrophage repolarization to the M1 phenotype. CRISPR-Cas9-mediated editing of the CD47 gene on tumor cells blocks antiphagocytic signals, enhancing M1 macrophage phagocytosis and increasing the antitumor immune response. This multimodal strategy triggers strong local antitumor effects and controls metastatic lesions via systemic immune activation, demonstrating significant potential for colorectal cancer treatment. Furthermore, this approach offers a new direction for overcoming resistance to immunotherapies and improving clinical outcomes.
研究领域:
肿瘤学
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免疫治疗
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纳米医学
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基因编辑
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癌症生物学
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肿瘤微环境
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AQUACULTURE
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2025
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IF 3.9
Tianyang Sun,Xinzhan Meng,Yaru Li,Hongquan Zhang,Ruitao He,Ruicong He,Tiegang Liu,Wenyao Lv,Xiaoyan Xu,Yubang Shen,Jianfeng Ren,Jiale Li
Aquaculture is critical for global food security, yet genetic improvement in farmed fish has progressed more slowly than in terrestrial livestock. Myostatin (MSTN), a conserved negative regulator of skeletal muscle growth in vertebrates, presents a prime target for enhancing aquaculture productivity. In this study, we employed CRISPR/Cas9 ribonucleoprotein (RNP) complexes to disrupt mstnb , the orthologous gene of livestock mstn , in grass carp ( Ctenopharyngodon idella ), the most economically important freshwater aquaculture species in China. Co-injection of five single-guide RNAs (sgRNAs) targeting mstnb exon 1 with Cas9 protein into single-cell embryos achieved 62.0–97.8 % editing efficiency in F 0 mosaic individuals, with an overall population efficiency of 67.1 %. At 7 months post-hatching, mstnb -knockout (KO) fish displayed 17.5 % greater body weight, 8.8 % increased body width, and 7.6 % higher body height compared to control siblings. Histological analysis revealed marked muscle fiber hypertrophy, with cross-sectional area increases of 9.0–26.3 % in cephalosome, trunk, and rump muscles. KO fish also displayed reduced hepatic lipid and glycogen accumulation, indicative of improved metabolic efficiency. While muscle amino acid composition remained largely unchanged, we observed a modest decrease in ω-3 polyunsaturated fatty acids (e.g., EPA and DHA). This study marks the first successful application of CRISPR/Cas9 in grass carp, demonstrating that mstnb disruption enhances growth and muscle development without substantially compromising meat quality. Our results underscore the potential of genome editing for sustainable aquaculture breeding, offering a promising strategy to address rising global protein demands.
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