ACS Nano
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2023
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IF 17.1
Dou Wang,Xuedong Wang,Feidi Ye,Jin Zou,Jiuxin Qu,Xingyu Jiang
Conventional nucleic acid detection technologies usually rely on amplification to improve sensitivity, which has drawbacks, such as amplification bias, complicated operation, high requirements for complex instruments, and aerosol pollution. To address these concerns, we developed an integrated assay for the enrichment and single molecule digital detection of nucleic acid based on a CRISPR/Cas13a and microwell array. In our design, magnetic beads capture and concentrate the target from a large volume of sample, which is 100 times larger than reported earlier. The target-induced CRISPR/Cas13a cutting reaction was then dispersed and limited to a million individual femtoliter-sized microwells, thereby enhancing the local signal intensity to achieve single-molecule detection. The limit of this assay for amplification-free detection of SARS-CoV-2 is 2 aM. The implementation of this study will establish a “sample-in-answer-out” single-RNA detection technology without amplification and improve the sensitivity and specificity while shortening the detection time. This research has broad prospects in clinical application.
研究领域:
生物技术
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分子生物学
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诊断方法
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高影响因子
JOURNAL OF NANOBIOTECHNOLOGY
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2023
·Vol. 21
·期 1
·1-8
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IF 10.2
Zheng Laibao,Jiang Yayun,Huang Fuyuan,Wu Qiaoli,Lou Yongliang
A multimodal analytical strategy utilizing different modalities to cross-validate each other, can effectively minimize false positives or negatives and ensure the accuracy of detection results. Herein, we establish a colorimetric, photothermal, and fluorescent triple modal CRISPR/Cas12a detection platform (CPF-CRISPR). An MNPs-ssDNA-HRP signal probe is designed to act as a substrate to trigger three signal outputs. In the presence of the DNA target, MNPs-ssDNA-HRP is cleaved by the activated CRISPR/Cas12a, resulting in the release of HRP and generating short DNA strands with 3-terminal hydroxyl on magnetic beads. The released HRP subsequently catalyzed TMB-H2O2 reaction and oxidized TMB is used for colorimetric and photothermal signal detection. Under the catalysis of terminal deoxynucleotidyl transferase (TdT), the remaining short DNA strands are used as primers to form poly-T and function as scaffolds to form copper nanoclusters for fluorescent signal output. To verify the practical application of CPF-CRISPR, we employed MRSA as a model. The results demonstrate the platform’s high accuracy and sensitivity, with a limit of detection of 101 CFU/mL when combined with recombinase polymerase amplification. Therefore, by harnessing the programmability of CRISPR/Cas12a, the biosensor has the potential to detect various drug-resistant bacteria, demonstrating significant practical applicability.
研究领域:
生物技术
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分子生物学
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分析化学
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微生物学
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Nature Protocols
·
2023
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IF 14.8
Liu Renmei,Yao Jing,Zhou Siyu,Yang Jing,Zhang Yaqiang,Yang Xiaoyan,Li Leshi,Zhang Yunbin,Zhuang Yingping,Yang Yi,Chen Xianjun
RNA molecules perform various crucial roles in diverse cellular processes, from translating genetic information to decoding the genome, regulating gene expression and catalyzing chemical reactions. RNA-binding proteins (RBPs) play an essential role in regulating the diverse behaviors and functions of RNA in live cells, but techniques for the spatiotemporal control of RBP activities and RNA functions are rarely reported yet highly desirable. We recently reported the development of LicV, a synthetic photoswitchable RBP that can bind to a specific RNA sequence in response to blue light irradiation. LicV has been used successfully for the optogenetic control of RNA localization, splicing, translation and stability, as well as for the photoswitchable regulation of transcription and genomic locus labeling. Compared to classical genetic or pharmacologic perturbations, LicV-based light-switchable effectors have the advantages of large dynamic range between dark and light conditions and submicron and millisecond spatiotemporal resolutions. In this protocol, we provide an easy, efficient and generalizable strategy for engineering photoswitchable RBPs for the spatiotemporal control of RNA metabolism. We also provide a detailed protocol for the conversion of a CRISPR–Cas system to optogenetic control. The protocols typically take 2–3 d, including transfection and results analysis. Most of this protocol is applicable to the development of novel LicV-based photoswitchable effectors for the optogenetic control of other RNA metabolisms and CRISPR–Cas functions.
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JOURNAL OF HAZARDOUS MATERIALS
·
2024
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IF 12.2
Manyan Qiu,Zhiyu Yuan,Nan Li,Xinyan Yang,Xianlong Zhang,Yujun Jiang,Qianyu Zhao,Chaoxin Man
Sensitive detection and point-of-care test of bacterial pathogens is of great significance in safeguarding the public health worldwide. Inspired by the characteristics of horseradish peroxidase (HRP), we synthesized a hybrid nanoflower with peroxidase-like activity via a three-component self-assembled strategy. Interestingly, the prepared nanozyme not only could act as an alternative to HRP for colorimetric biosensing, but also function as a unique signal probe that could be recognized by a pregnancy test strip. By combining the bifunctional properties of hybrid nanoflower, isothermal amplification of LAMP, and the specific recognition and non-specific cleavage properties of CRISPR/Cas12a system, the dual-readout CRISPR/Cas12a biosensor was developed for sensitive and rapid detection of Salmonella enterica . Moreover, this platform in the detection of Salmonella enterica had limits of detection of 1 cfu/mL (colorimetric assay) in the linear range of 10 1 –10 8 cfu/mL and 10 2 cfu/mL (lateral flow assay) in the linear range of 10 2 –10 8 cfu/mL, respectively. Furthermore, the developed biosensor exhibited good recoveries in the spiked samples (lake water and milk) with varying concentrations of Salmonella enterica . This work provides new insights for the design of multifunctional nanozyme and the development of innovative dual-readout CRISPR/Cas system-based biosensing platform for the detection of pathogens. Environmental implication The presence of bacterial pathogens is pervasive in the environment and poses a significant threat to public health safety. To prevent foodborne disease outbreaks, it is crucial to develop sensitive and reliable pathogen detection strategies for rapid and on-site detection of pathogens in both environmental and food samples. Herein, we developed a novel dual-readout CRISPR/Cas biosensing platform based on nanozyme with bifunctional properties and loop-mediated isothermal amplification (LAMP) for highly sensitive and on-site detection of Salmonella enterica in lake water and milk samples. The proposed method has good stability and high accuracy in the detection of Salmonella enterica .
研究领域:
生物技术
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纳米技术
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病原体检测
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生物传感器
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即时诊断
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TALANTA
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2025
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IF 6.1
Tao Meng,Qi Kang,Jiashuo Xu,Shuang Zhao,Tianqi Liu,Dianming Zhou,Xiaoqun Gong,Jianyu Zhang
The CRISPR/Cas12a system has become a powerful tool in biosensing because of its specific target recognition ability and highly efficient trans -cleavage activity. However, a problem faced by the CRISPR/Cas12a system when directly used for trace detection is the linear amplification efficiency of single-cycle digestion. Here, we present a novel hairpin reporter-driven CRISPR/Cas12a (HR-CRISPR) amplification system that establishes a positive feedback loop within the CRISPR/Cas12a platform to finish an exponential and sensitive signal amplification in a one-step reaction. As proof of concept, we applied this strategy to the terminal deoxynucleotidyl transferase (TdT) activity assay without pre-amplification procedure. The polyT strand extended by TdT hybridizes with crRNA, activating Cas12a, which then cleaves the FQ-hairpin reporter. The cleavage products are further elongated by reverse transcriptase using crRNA as a template, reactivating Cas12a and producing exponentially amplified fluorescence signals. This assay offers a simple yet highly sensitive approach for quantifying TdT activity, achieving a low detection limit of 4.55 × 10 −6 U. Moreover, it is applicable for inhibitor screening and monitoring TdT activity in human serum samples.
研究领域:
分子生物学
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生物技术
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医学检测
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高影响因子
NUCLEIC ACIDS RESEARCH
·
2025
·Vol. 53
·期 7
·
IF 13.1
Liu Qiong,Jiang Zhou,Li Sheng,Li Yinfeng,Wan Yingfei,Hu Zhenyu,Ma Shimeng,Zou Zhen,Yang Ronghua
Accurate identification of single-nucleotide variants (SNVs) is critical in clinical diagnostics but remains challenging due to subtle free energy variations, particularly for hard-to-detect SNVs such as wobble base pairs and those in high guanine–cytosine (GC) regions. Here we report a high-energetic-penalty SNV detection (HEPSD) platform that redesigns the hybridization regions of CRISPR RNA (crRNA) in the CRISPR/Cas12a system. This system employs a binary crRNA architecture design that enables the activation of the cleavage activity of Cas12a while amplifying the energetic penalty for single-nucleotide mismatches through nonequilibrium hybridization-driven regulation. Consequently, the entire targeting region of CRISPR/Cas exhibits a marked preference for mutations in genomic DNA, while preventing false activation induced by sequences containing a single mismatched nucleotide. Moreover, HEPSD exhibits exceptional differentiation performance for hard-to-detect SNVs including wobble mutations at extreme GC contents. As proof of principle, profiling of BRAF V600E and EGFR L858R tumor mutations down to a 0.01% variant allele frequency was achieved, enabling accurate discrimination of 132 clinical sample pairs, which showed high consistency with quantitative polymerase chain reaction-based techniques and next-generation sequencing. The proven effectiveness of this platform showcases its potential for clinical molecular diagnostics and expands the fundamental scope of hybridization-based protocols.
研究领域:
分子生物学
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医学诊断
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基因组学
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INFECTION GENETICS AND EVOLUTION
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2015
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IF 3.015
Qinqin Huang,Hongping Luo,Minqiang Liu,Jie Zeng,Abualgasim Elgaili Abdalla,Xiangke Duan,Qiming Li,Jianping Xie
Clustered regularly interspaced short palindromic repeats (CRISPR) are present in the genome of 40% bacteria and 90% archaea . CRISPR and accompanying Cas proteins constitute an adaptive immune system against disruptive mobile genetic elements . Two CRISPRs and 9 genes encoding CRISPR-associated proteins have been found in the genome of Mycobacterium tuberculosis . The CRISPR-associated Cas2 is an endoribonuclease required for the acquisition of new spacers. In this study, Cas2 encoded by Rv2816c was expressed in Mycobacterium smegmatis lacking CRISPR-Cas system and its role in stress responses of M. smegmatis in vitro and within macrophages was studied. We found that Cas2 mediated M. smegmatis stress response changes were associated with the altered expression of sigma factors which involved in mycobacterial stress response and virulence. We also found that Cas2 decreased the survival of M. smegmatis within macrophages. This study provides new insights on the role of Cas2.
研究领域:
微生物学
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免疫学
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分子生物学
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高影响因子
mSphere
·
2025
·Vol. 10
·期 6
·
IF 3.1
Jieying Li,Yuxiao Liu,Jingsi Jiang,Fang Chen,Nan Zhang,Xun Kang,Lin Liu,Yingjuan Wang,Qianfeng Xia,Chuanlong Zhu,Dai Kuang
Klebsiella pneumoniae is a globally recognized microbial pathogen with significant clinical impact. The bacterium harbors the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems, which provide adaptive immunity against invading foreign nucleic acids. Recent studies suggest that certain CRISPR-Cas systems can regulate endogenous genes, influencing bacterial virulence. However, their role in regulating pathogenicity in K. pneumoniae remains poorly understood. This study investigates the regulatory role of the type I-E* CRISPR-Cas system in a hypervirulent K. pneumoniae strain, focusing on its impact on histidine metabolism and pathogenicity. Transcriptome analyses identified differentially expressed genes (DEGs) between the casABECD-deletion and wild-type strains, including significant upregulation of the histidine utilization (Hut) operon and downregulation of biofilm-related genes. These molecular changes resulted in enhanced histidine metabolic activity, reduced biofilm formation, attenuated virulence in A549 lung epithelial cells, and improved survival of Galleria mellonella, as validated through phenotypic and virulence assays. Our bioinformatic analysis indicated that the CRISPR-Cas system in K. pneumoniae targets the hutT sequence, which is part of the Hut operon. Furthermore, the overexpression of hutT mitigated CRISPR-Cas-mediated repression of the Hut operon, as observed in virulence assays, while simultaneous deletion of hutH and casABECD restored the reduced virulence in the ΔcasABECD strain. Additionally, deletion of casABECD significantly enhances the growth of the strain in medium with histidine as the sole carbon source, highlighting the intricate regulatory role of the CRISPR-Cas system in metabolic adaptation. Collectively, these findings uncover a novel role for the CRISPR-Cas system in regulating metabolic pathways and virulence in hypervirulent K. pneumoniae.
研究领域:
微生物学
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分子遗传学
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传染病学
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细菌致病机制
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CRISPR生物学
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CHEMICAL ENGINEERING JOURNAL
·
2025
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IF 13.2
Hongkai Wang,Yinzheng Niu,Hui Liu,Ye Zhu
Nucleic acid detection is crucial for early disease diagnosis and related clinical applications. Exploring nucleic acid amplification-free yet highly sensitive detection strategies is of significance for the development of accurate and user-friendly point-of-care testing devices. Herein, a modularized universal electrochemical sensing strategy was proposed based on the synergistic effect of CRISPR/Cas12 system and nanoporous materials. The trans -cleavage activity of Cas12a, which was activated by HPV-16 target, was utilized to cut the gatekeepers of mesoporous silica nanoparticle nanocontainers. The released methylene blue was entrapped by a mercaptobenzoic acid functionalized nanoporous gold (NPG) sensing platform. By virtue of the excellent electrocatalytic ability of NPG, ultrasensitive detection of HPV-16 was achieved over a broad linear range of 7 orders of magnitude with a detection limit as low as 0.41 fM. Moreover, determination of HPV-18 was easily achieved by only altering crRNA, revealing the remarkable universality of this strategy and its superiority of modularized design. The practical applicability of the proposed sensing strategy was verified in terms of specificity, stability, and complex biological sample analysis, demonstrating its promising prospects in the development of reliable, universal, robust, and user-friendly point-of-care diagnostic devices.
研究领域:
生物技术
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医学诊断
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纳米科学
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DOI
高影响因子
EUROPEAN JOURNAL OF MEDICAL RESEARCH
·
2024
·
IF 2.8
Lu Yan,Yin-Sen Song,Jian Zhou,Lin Zhu,Tian-Wei Shi,Hui-Qian Yu,Zi-Qing Dong,Wei Wang,Ting Long,Hao-Ying Liu,Zhe-Ye Shi,Jian-Guo Li
Background Nicastrin, a subunit of the γ-secretase complex, is encoded by the NCSTN gene and regulates notch signaling, it is involved in the pathogenesis of hidradenitis suppurativa (HS), Alzheimer disease (AD), and liver cancer. However, the animal models for studying HS are relatively scarce. Methods CRISPR/Cas-mediated genetic engineering was used to generate targeted knockout offspring mice (C57BL/6J). Different doses (10 mg/kg, 20 mg/kg, and 30 mg/kg) and injection methods (subcutaneous/intraperitoneal/gavage injection) of tamoxifen were used to induce the construction of NCSTN knockout mice (mice model). The expressions of nicastrin, NICD1, hes1 in skin, brain, and liver tissue in mice model and wild-type (WT) mice were measured by qRT-PCR and IHC. Results The construction of mice model was successfully induced by tamoxifen, knockout efficiency was 93%, there was no difference in knockout efficiency among three doses, injection methods, genders ( P > 0.05). HS-like lesions appeared on the skin of NCSTN knockout mice after 1 month of treatment with tamoxifen, male mice had a higher number of skin lesions compared to female mice (male vs female = 76.5% vs 41.7%, P = 0.027). Compared with WT mice, the expressions of nicastrin (skin P = 0.0009, brain P = 0.0194, liver P = 0.0066), NICD1 (skin P = 0.0115, brain P = 0.0307, liver P = 0.008), hes1 (skin P = 0.0476, brain P = 0.0143, liver P = 0.0003) in mice model all decreased. Conclusions The NCSTN knockout mouse might be employed as HS animal model; Reducing nicastrin may affect the expression of notch1–hes1 pathway molecules in skin, brain, and liver tissues; low dose (10 mg/kg/d) tamoxifen could be used to induce the deletion of the target gene in mice.
研究领域:
分子生物学
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遗传学
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病理学
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肿瘤学
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PubMed
高影响因子
ACS Sensors
·
2025
·
IF 9.1
Lingxuan Qu,Lingyi Meng,Xilin Sun,Wei Cui,Jin Shi,Yuqi Wang,Qingshuang Hu,Shuping Xu,Bingxin Sun,Chongyang Liang
Aiming at realizing field detection during HIV-1 patient screening in developing countries and point of care testing (POCT) of virus levels in HIV-1 patients receiving medication, an urgent demand for portable nucleic acid detection technology with low cost and sensitivity is raised. To solve this, a CRISPR/Cas13a-based electrochemical detection platform by a multiple combined crRNAs strategy is developed. This sensing platform is based on the Ion Current Rectification regulation through a Porous Anodic Alumina membrane decorated with ssRNA chains, which are trans-cleavage substrates for activated Cas13a and become shorter when the target gene exists, resulting in an altered motion of transmembrane ions. This platform was proven to be feasible and ultrasensitive for tracing the HIV-1 virus by multiple applicable crRNAs combination, and its clinical sample detection limit arrives at 60 copies/μL for HIV-1 patients’ serum samples. Importantly, this method is amplification-free, and its sensitivity is beyond the nonamplification fluorescence-based nucleic acid strategy by one order of magnitude. This sensor is facile to use and cost-effective, and can also be extended to other RNA-based pathogenic microorganism detections. This POCT method facilitates self-monitoring of drug resistance in HIV-1 patients, regardless of medication status.
研究领域:
生物传感器
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分子诊断
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病毒学
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生物电子学
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即时检测
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CRISPR技术
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纳米技术
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RNA检测
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ANALYTICAL CHEMISTRY
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2025
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IF 6.7
Muhammad Sohail,Shuyao Ma,Barira Mushtaq,Muhammad Usman Haider,Bingzhi Li,Xing Zhang,He Huang
Conventional CRISPR/Cas sensing platforms exhibit poor efficiency concerning reporter-based demerits, including their interference-labile nature, photobleaching, low robustness due to a single output signal, and probe-concentration dependence. Herein, a carbon dots (CDs)-based dual-emissive fluorescent ratiometric CRISPR/Cas reporter platform was fabricated for biosensing and other analytical applications to bottleneck the demerits of conventional reporters, integrating the benefits of a ratiometric strategy and four-element doped carbon dots (4D CDs) as a transducer. Briefly, doping enhances the optical and physicochemical traits of CDs and minimizes the effect of the interfering species. A series of state-of-the-art N, P, S, and Cu codoped CDs (4D CDs) were synthesized using the hydrothermal approach and statistical tools, such as Box-Behnken design, analysis of variance, and others, enhancing photophysical traits, surface features, and sensitivity of CDs. The red-emissive CDs were prepared by using the same procedure but different precursors. The optimum 4D CDs (blue-emissive) and red-emissive CDs were used to unleash the principle of the fluorescent ratiometric CRISPR/Cas reporter system for diverse applications. Finally, the designed 4D CDs-based CRISPR/Cas biosensor was applied for nucleic acid monitoring, such as the COVID-19 nucleic acid. This project disclosed the controlled-doping principle to synthesize 4D CDs and unleashed the mechanism of ratiometric dual-emissive CRISPR/Cas-powered reporters for precise sensing applications. We anticipate the implementation of this technology in commercial analytical, biosensing, point-of-care, and other applications.
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