Microbial Cell Factories
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2022
·Vol. 21
·期 1
·1-13
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IF 6.352
Xin Qinglong,Chen Yudan,Chen Qianlin,Wang Bin,Pan Li
Background Bacillus amyloliquefaciens is generally recognized as food safe (GRAS) microbial host and important enzyme-producing strain in the industry. B.amyloliquefaciens LB1ba02 is a production strain suitable for secreting mesophilic α-amylase in the industry. Nevertheless, due to the low transformation efficiency and restriction-modification system, the development of its CRISPR tool lags far behind other species and strains from the genus Bacillus . This work was undertaken to develop a fast and efficient gene-editing tool in B.amyloliquefaciens LB1ba02. Results In this study, we fused the nuclease-deficient mutant Cas9n (D10A) of Cas9 with activation-induced cytidine deaminase (AID) and developed a fast and efficient base editing system for the first time in B. amyloliquefaciens LB1ba02. The system was verified by inactivating the pyrF gene coding orotidine 5'-phosphate decarboxylase and the mutant could grow normally on M9 medium supplemented with 5-fluoroorotic acid (5-FOA) and uridine (U). Our base editing system has a 6nt editing window consisting of an all-in-one temperature-sensitive plasmid that facilitates multiple rounds of genome engineering in B. amyloliquefaciens LB1ba02. The total editing efficiency of this method reached 100% and it achieved simultaneous editing of three loci with an efficiency of 53.3%. In addition, based on the base editing CRISPR/Cas9n-AID system, we also developed a single plasmid CRISPR/Cas9n system suitable for rapid gene knockout and integration. The knockout efficiency for a single gene reached 93%. Finally, we generated 4 genes ( aprE , nprE , wprA , and bamHIR ) mutant strain, LB1ba02△4. The mutant strain secreted 1.25-fold more α-amylase into the medium than the wild-type strain. Conclusions The CRISPR/Cas9n-AID and CRISPR/Cas9n systems developed in this work proved to be a fast and efficient genetic manipulation tool in a restriction-modification system and poorly transformable strain.
研究领域:
微生物学
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基因工程
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生物技术
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DOI
PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
·
2024
·
IF 8
Zhina Wu,Yingtong Wang,Yaqin Zhang,Jingzheng Yi,Yutong Li,Jiasi Wang,Min Hu,Di Wang
Periodontitis is one of the most prevalent oral diseases, and the diagnosis and monitoring of periodontitis rely on clinical and imaging examinations. Matrix metalloproteinase-8 (MMP-8) in saliva is a reliable indicator of periodontitis. The traditional detection method is enzyme-linked immunosorbent assay (ELISA), which requires professional expertise and is not sensitive enough to low-abundance protein detection. In this study, we developed a nanoimmunoassay combined with clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated protein (Cas)12a for ultrasensitive detection of MMP-8 in saliva. Antibody-DNA-gold nanoparticles (AuNPs) and trans-cleavage activity of CRISPR/Cas12a were employed for signal amplification. With this protocol, the limit of detection (LoD) for MMP-8 was reduced to the fg/mL, indicating 100-fold higher sensitivity than that of the commercial ELISA kit and its amenability for ultrasensitive detection of trace proteins. The diagnostic sensitivity of the assay was 100 %, and the specificity was 83.3 % based on the testing of 23 clinical saliva samples. The sensitivity and specificity were considerably higher than those for ELISA. Thus, this immuno-CRISPR/Cas12a assay holds promise for both diagnosing and monitoring periodontitis in clinical settings.
研究领域:
口腔科学
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分子生物学
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生物技术
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临床诊断
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产品关联
DOI
高影响因子
Synthetic and Systems Biotechnology
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2019
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IF 0
Jingjun Mo,Shuwen Wang,Wan Zhang,Chunyu Li,Zixin Deng,Lixin Zhang,Xudong Qu
Actinobacteria able to produce varieties of bioactive natural products have been long appreciated by the field of drug discovery and development. Recently, a few of CRISPR/Cas9 systems bearing different types of replicons (pSG5 and pIJ101) were developed to efficiently edit their genomes. Despite wide application in gene editing, their utility in editing challenging DNA regions e.g. high sequence identity has not been compared. In this study, we confirmed that the widely used temperature-sensitive pSG5 replicon is indeed not suitable for editing modular polyketide synthase (PKS) genes due to causing unpredicted gene recombination. This problem can be addressed by replacing the pSG5 with the segregationally unstable pIJ101 replicon. By introducing a counter-selection marker CodA, convenient cloning sites in the single guide RNAs (sgRNAs) and homologous template scaffolds, we developed a new CRISPR-Cas9 system pMWCas9. This system was successfully used to delete/replace erythromycin PKS and other biosynthetic genes in Saccharopolyspora erythraea and Streptomyces sp. AL2110. By swapping the promoters of antB and antC with ermE and kasOp , we achieved a deacyl-antimycin hyper producer which produces a 9-fold higher yield than the original Streptomyces sp. AL2110 strain. Our results provide a robust and useful Cas9 tool for genetic studies in Actinobacteria .
研究领域:
遗传学
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微生物学
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生物技术
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药物发现
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DOI
PubMed
MICROCHEMICAL JOURNAL
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2025
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IF 5.1
Jiayi Zhang,Zichun Song,Jiuying Cui,Wenbo Li,Song Huang,Bo Ling,Kai Zhang,Xianjiu Liao,Jihua Wei
The human gastrointestinal system contains the conditional pathogen Escherichia coli ( E. coli ), which is a significant cause of vomiting and diarrhea—both of which can be fatal in extreme cases. Given the surge in hospital infections and water pollution, the development of early detection and diagnosis procedures is imperative. This study proposes an electrochemical biosensor for E. coli detection, utilizing nucleic acid aptamers as recognition elements and using E. coli-2571 as an example. It is based on the CRISPR/Cas gene-editing technology. Specifically, the collateral cleavage activity of Cas12a shifts the electrochemical signal after loading the DNA sequence tagged with an electrochemical signal onto the gold electrode, which ultimately enables the identification of E. coli . The biosensor has a detection limit of 13.1 CFU/mL and exhibits a good linear relationship in the concentration range of 10 2 ––10 7 CFU/mL. It was effectively used for the quantitative detection of E. coli in milk and pure water, with a recovery rate ranging from 93.12 % to 98.93 %. The biosensor offers a rapid, precise, affordable, and sensitive tool for E. coli detection. It is anticipated to become a powerful instrument for identifying microorganisms that pose a risk to public health and human health.
研究领域:
分子生物学
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微生物学
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生物传感技术
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公共健康
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产品关联
DOI
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
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2025
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IF 7.7
Yue Wang,Shuning Lei,Youjun Zeng,Wei Ni,Jie Zhou,Guo-Jun Zhang
MicroRNA(miRNA) has emerged as a particularly promising biomarker for the early diagnosis of Alzheimer's disease (AD). However, current detection methods for miRNA remain challenges in terms of sensitivity and pre-amplification steps. This study proposes a novel surface plasmon resonance imaging (SPRi) biosensor that combines tetrahedral DNA nanostructures (TDN)-based surface organization with CRISPR-Cas13a-mediated molecular recognition, enabling amplification-free, sensitive, and specific detection of AD-related miRNA-137. The biotin-labeled RNA reporters are anchored to TDN framework on the gold film surface, ensuring the orientation of the probe. In the presence of target miRNA-137, the CRISPR-Cas13a system activates, cleaves these reporters and prevents subsequent binding of streptavidin-HRP, thereby inhibiting precipitate formation from the enzymatic reaction. Under optimal condition, this sensor, integrated with the self-developed highly sensitive intensity-modulated SPRi system, displays a good linear response to miRNA-137 ranging from 0.5 pM to 500 nM with a detection limit of 212 fM. The platform demonstrates excellent specificity of discriminating single-base mutation, and reliable analysis capability in clinical plasma samples. Notably, our sensor confirms significantly reduced miRNA-137 levels in AD patients compared to healthy people. This innovative assay provides a powerful tool for the detection of AD-related miRNAs, promising to advance early diagnosis research for AD.
研究领域:
分子诊断
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神经生物学
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生物传感技术
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纳米医学
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转化医学
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分子生物学
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临床检验
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产品关联
DOI
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
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2025
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IF 7.7
Zhi-Li Yao,Qing-Nan Li,Yong-Ji Li,Hao-Rui Li,Si-Wei Yang,Chen-Yu Wang,Jia-Yi Shi,Shun-Li Wu,Shuang-Qing Liu,Gui-Mei Han,Jing Wang,Li-Yong Xing,Yan Zhao,De-Ming Kong,Qi-Liang Cai
Clear cell renal cell carcinoma (ccRCC) is the most prevalent form of kidney cancer in adults, with rising global incidence and mortality rates. Early diagnosis is critical for improving patient outcomes. However, current diagnostic techniques often rely on expensive and complex instrumentation, which limits their accessibility. This creates an urgent need for affordable, sensitive, and user-friendly diagnostic methods. MiR-210–3p, a biomarker significantly upregulated in ccRCC, shows promise for non-invasive diagnostic applications. In this study, we innovatively developed a fluorescence-colorimetric dual-mode platform for miR-210–3p detection by utilizing a recently identified hemin aptamer with a stem-loop structure and by combining the trans -cleavage activity of the CRISPR/Cas12a system with the peroxidase-like activity of the aptamer/hemin complex. Moreover, using fluorescently labeled aptamer or its complementary sequence as cleavage substrates for CRISPR/Cas12a allows us to achieve two types of dual-mode detection: fluorescence “turn on” combined with colorimetric “turn off”, and fluorescence “turn on” combined with colorimetric “turn on”. This dual-mode detection platform combines sensitive quantification (with a detection limit for miR-210-3p of 0.95 pM) with rapid and visually interpretable on-site analysis, significantly enhancing the reliability and accuracy of test results. The platform has good scalability, as it is capable of detecting miR-210-3p and can also be extended to detect other tumor biomarkers. This versatility holds great potential for integration with big data analytics to personalize miRNA profiles, aiding in tumor staging, monitoring, and the development of treatment strategies, thereby advancing the field of precision medicine.
研究领域:
分子生物学
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肿瘤学
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诊断学
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产品关联
DOI
高影响因子
BIOSENSORS & BIOELECTRONICS
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2024
·
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
SENSORS AND ACTUATORS B-CHEMICAL
·
2020
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IF 7.1
Xianfeng Wang,Xiaolong Chen,Chengxiang Chu,Yuanyi Deng,Mei Yang,Zhong Ji,Faliang Xu,Danqun Huo,Yang Luo,Changjun Hou
High-performance detection of adenosine triphosphate (ATP) is of great importance due to its irreplaceable roles in various physiological processes. In this work, a four-stage signal amplification was established for ATP detection based on an allosteric probe-conjugated strand displacement amplification (SDA) integrated with CRISPR/Cpf1 system (ASD-Cpf1). An allosteric probe (AH) with a hairpin structure was designed to reduce background interference from self-extension and non-specific amplification during the SDA reaction. ATP bound with the aptamer segment at 5′-terminal of AH probe and subsequently opened the AH probe to activate the SDA reaction. The amplification products were recognized by CRISPR/Cpf1 system and triggered the ssDNase activity of Cpf1, resulting in robust cleavage of surrounding DNA FQ-reporter (fluorophore-quencher labeled short single-stranded DNA sequence). With the four-step amplification, ATP was measured with high sensitivity and low background interference. Under the optimal conditions, a linear relationship was obtained between the fluorescence response and ATP concentrations ranging from 2 pM to 10 μM with a limit of detection (LOD) as low as 1.8 pM. Successful detection of ATP in diluted human serum samples further indicated that ASD-Cpf1 system provided a potential platform for detection of small molecules.
研究领域:
分析化学
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分子生物学
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生物技术
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产品关联
DOI
高影响因子
Cell Reports
·
2025
·
IF 6.9
Tianzi Wei,Jiaxuan Li,Xiang Lei,Risheng Lin,Qingyan Wu,Zhenfeng Zhang,Shimin Shuai,Ruilin Tian
Adenosine-to-inosine (A-to-I) RNA editing is a critical post-transcriptional modification that diversifies the transcriptome and influences various cellular processes, yet its regulatory mechanisms remain largely unknown. Here, we present two complementary CRISPR-based genetic screening platforms: CREDITS (CRISPR-based RNA editing regulator screening), which enables genome-scale identification of editing regulators using an RNA recorder-based reporter system, and scCREDIT-seq (single-cell CRISPR-based RNA editing sequencing), which provides multiplexed single-cell characterization of transcriptome and editome changes for pooled perturbations. By screening 1,350 RNA-binding proteins, we identified a series of A-to-I editing regulators. Mechanistic investigation revealed DDX39B as a global repressor of A-to-I editing, which functions by preventing double-stranded RNA accumulation through its helicase activity. Targeting DDX39B significantly enhances the efficiency of RNA-editing-based tools, such as CellREADR (cell access through RNA sensing by endogenous ADAR) and LEAPER (leveraging endogenous ADAR for programmable editing of RNA), and disrupts hepatitis D virus (HDV) RNA editing homeostasis. These technological advances not only expand our understanding of RNA editing regulation but also provide powerful tools for exploring tissue-specific and context-dependent RNA modification mechanisms, with broad implications for therapeutic development.
研究领域:
分子生物学
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遗传学
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RNA生物学
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CRISPR技术
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病毒学
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基因编辑
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功能基因组学
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产品关联
DOI
PubMed
高影响因子
Biology-Basel
·
2021
·Vol. 10
·期 10
·1038
·
IF 5.079
Haodong Cui,Min Jiang,Wenhua Zhou,Ming Gao,Rui He,Yifan Huang,Paul K. Chu,Xue-Feng Yu
Simple SummaryCRISPR/Cas9 system as a potential gene editing platform has been widely applied in biological engineering and disease therapies. To achieve precise gene targeting, active CRISPR/Cas9 components must be efficiently transported to targeted cells. As a simple and effective strategy, Cold Atmospheric Plasma (CAP) treatment has been demonstrated for the transmembrane delivery of various exogenous materials. In comparison with carrier-dependent delivery methods, this carrier-free platform provides a promising alternative to circumvent the obstacles of biosafety and complicated preparation processes. In this work, a CAP-based CRISPR/Cas9 carrier-free delivery platform has been established and corresponding mechanism related to efficient transportation has been explored. Briefly, the efficient production of bioactive species in culture media after CAP treatment alters cell membrane potential and permeability, which facilitates cytosolic delivery of active CRISPR/Cas9 components via passive diffusion and ATP-dependent endocytosis pathways, resulting in efficient genome editing and gene silencing. This carrier-free strategy using CAP-based transportation may also be extended to other active biomolecules in drug delivery and gene therapy.AbstractA carrier-free CRISPR/Cas9 ribonucleoprotein delivery strategy for genome editing mediated by a cold atmospheric plasma (CAP) is described. The CAP is promising in many biomedical applications due to efficient production of bioactive ionized species. The MCF-7 cancer cells after CAP exposure exhibit increased extracellular reactive oxygen and nitrogen species (RONS) and altered membrane potential and permeability. Hence, transmembrane transport of Ca2+into the cells increases and accelerates ATP hydrolysis, resulting in enhanced ATP-dependent endocytosis. Afterwards, the increased Ca2+and ATP contents promote the release of cargo into cytoplasm due to the enhanced endosomal escape. The increased membrane permeability also facilitates passive diffusion of foreign species across the membrane into the cytosol. After CAP exposure, the MCF-7 cells incubated with Cas9 ribonucleoprotein (Cas9-sgRNA complex, Cas9sg) with a size of about 15 nm show 88.9% uptake efficiency and 65.9% nuclear import efficiency via passive diffusion and ATP-dependent endocytosis pathways. The efficient transportation of active Cas9sg after the CAP treatment leads to 21.7% and 30.2% indel efficiencies in HEK293T and MCF-7 cells, respectively. This CAP-mediated transportation process provides a simple and robust alternative for the delivery of active CRISPR/Cas9 ribonucleoprotein. Additionally, the technique can be extended to other macro-biomolecules and nanomaterials to cater to different biomedical applications.Keywords:cold atmospheric plasma;CRISPR/Cas9;carrier-free transport;genome editing
研究领域:
生物医学工程
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遗传学
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分子生物学
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治疗学
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产品关联
DOI
PubMed
高影响因子
Advanced Science
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2025
·
IF 14.1
Shuai Wu,Yincheng Liu,Tianyu Zeng,Tianci Zhou,Yanting Sun,Ying Deng,Juan Zhang,Genxi Li,Yongmei Yin
Continued development of clustered regularly interspaced short palindromic repeats (CRISPR)-powered biosensing system on the electrochemical interface is vital for accurate and timely diagnosis in clinical practice. Herein, an electrochemical biosensor based on manganese metal-organic frameworks (MOFs)-enhanced CRISPR (MME-CRISPR) is proposed that enables the efficient detection of circulating tumor DNA (ctDNA). In this design, customized enzyme stimulants (Mn 2+ ) are co-assembled with Cas12a/crRNA to form enzyme-MOF composites, which can be released quickly under mild conditions. The MOFs-induced proximity effect can continuously provide adequate Mn 2+ to sufficiently interact with Cas12a/crRNA during the release process, enhancing the trans-cleavage activity of complex available for biosensor construction. The MOFs-based enzyme biocomposites also afford efficient protection against various external stimulus. It is demonstrated that the developed biosensor can achieve ultrasensitive detection of epidermal growth factor receptor L858R mutation in ctDNA with a low detection limit of 0.28 f m without pre-amplification. Furthermore, the engineered mismatch crRNA enables the biosensor based on MME-CRISPR to detect single nucleotide variant with a high signal-to-noise ratio. More importantly, it has been successfully used to detect the targets in clinical practice, requiring low-dose samples and a short time. This strategy is believed to shed new light on the applications of cancer diagnosis, treatment, and surveillance.
研究领域:
生物医学工程
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分子诊断学
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纳米医学
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产品关联
DOI
PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
·
2024
·
IF 8
Sirui Liu,Xinlan Bian,Furong Zhao,Min Cao,Zhaoyang Shang,Bingzhi Li,Xing Zhang
High mannose is overexpressed on the cell surface during carcinogenesis; however, there are currently no efficient techniques for its detection. Herein, we propose an assay called RADAR ( r emote high m a nnose d etection on cell surf a ce with C R ISPR) for the quantification of high mannose, leveraging the integration of high mannose-binding aptamer and CRISPR technique. The high mannose-binding aptamer is hybridized with a locker, which is released upon specific binding to high mannose. The released locker then activates CRISPR/Cas12a to produce a fluorescence signal. The "remote" concept in RADAR refers to the recognition of high mannose occurring on the cell surface, while signal generation takes place in a solution separate from the target. This feature makes RADAR suitable for the detection of suspended bio-samples without the need for sophisticated imaging. RADAR can detect high mannose Man 9 with a limit of detection of 2.5 nM, distinguish cancer cells from non-cancerous cells, and profile high mannose level in human serum for potential non-small cell lung cancer (NSCLC) diagnosis. This work represents the first attempt to integrate CRISPR technology into the aptasensing of high mannose, which may serve as a synthetic biology-enabled tool in glycochemistry and glycobiology.
研究领域:
合成生物学
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糖化学
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糖生物学
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生物技术
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癌症诊断
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产品关联
DOI
高影响因子
危险品化学品经营许可证（带存储）