NUCLEIC ACIDS RESEARCH
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2026
·Vol. 54
·期 5
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IF 15
Wang Zhongzhong,Wu Yi,Wang Zixuan,Zhang Simeng,Liu Hao,Nie You,Chen Kexin,Huang Yanjun,Zhou Yu,Cao Yuanyuan,Sun Lin,Hao Rongzhang
CRISPR–Cas12a has transformative potential in molecular diagnostics owing to its robust signal amplification, but its sustained activity state severely limits temporal programmability and precise nuclease control in complex detection workflows. Here, we demonstrate that the conserved crRNA scaffold secondary structure itself can be repurposed as a reversible and programmable conformational switch to regulate Cas12a activity. By introducing short complementary DNA blockers of tunable length, we achieved length-dependent disruption and remodeling of scaffold secondary structure, shifting LbCas12a into an inactive conformation. Scaffold structure was subsequently reinstated through either single or cooperative strand displacement activation, enabling time-resolved and on-demand restoration of Cas12a activity. The conserved scaffold ensures intrinsic assay universality, while its programmable rewiring markedly improves SNVs discrimination and enables compatibility with one-pot isothermal amplification assays, delivering analytical sensitivity comparable to conventional two-step assays. This regulatory framework was further demonstrated in the detection of Klebsiella pneumoniae and Mycobacterium tuberculosis. By validating the crRNA scaffold as a practical and programmable switch for Cas12a activity control, this work establishes a universal and reversible framework for scaffold rewiring to modulate CRISPR nucleases and offers mechanistic insight to guide future assay engineering.
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MICROCHEMICAL JOURNAL
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2023
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IF 4.8
Si Chen,Rong Fang,Yi Li,Fei Deng,Xing Liu,Danting Yang
Ochratoxin A (OTA) is classified as a Class 2B carcinogens and poses a significant risk bto the liver, kidney and other organs, potentially leading to carcinogenic and teratogenic effects. Accurate monitoring of OTA content in food is crucial for safeguarding human and animal health. Here, a novel approach called the C RISPR/Cas12a enzyme- l inked a ptamer- s orbent a ssay (CLASA) was developed, which integrates the high-performance trans -cleavage ability of CRISPR/Cas12a with the cost-effective and high-affinity properties of aptamer for ultra-sensitive OTA detection. In this strategy, we incubated aptamers on microwell plates, and used the fluorescence intensity changes caused by OTA and crRNA competitively bind OTA-specific aptamer to achieve the quantitative detection. Our sensitive CLASA method not only solves the problems of poor repeatability and relatively low sensitivity of fluorescence based-CRISPR/Cas12a reaction in solution, but also solves the defects of expensive antibody and false positive result in traditional ELISA method, achieving a limit of detection (LOD) as low as 171 fg·mL −1 with a broad linear range from 1 pg·mL −1 to 1000 pg·mL −1 . The presented CLASA approach was validated successfully by using commercial ELISA kit on rice and oat samples as well. Furthermore, by strategically modifying the sequences of aptamer and crRNA, our CLASA platform can be flexibly customized to detect a wide range of small molecular targets. This adaptability allows for the extension of our framework beyond nucleic acid substances, enabling the detection of non-nucleic acid compounds using the CRISPR-Cas12a system. As a result, our CLASA approach holds great promise as a versatile sensing kit with applications in various fields requiring sensitive and specific detection of diverse analytes.
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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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SENSORS AND ACTUATORS B-CHEMICAL
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2025
·
IF 7.7
Decai Yuan,Cheng Qiu,Hui Chen,Feng Liu,Shanshan Feng,Peiyi Zhang,Ying Qin,Tingting Fan,Yan Chen,Yuyang Jiang
Colorectal cancer (CRC) represents a significant global health issue, necessitating innovative approaches for early screening and diagnosis. Recent advances in molecular diagnostics have highlighted the potential of aptamers for use as highly specific and sensitive probes for detecting cancer biomarkers. In this study, we focus on the identification of aptamers that selectively bind to Solobacterium moorei (S. moorei) , a bacterium associated with CRC. By integrating these aptamers into electrochemical sensor platforms, a reliable diagnostic tool is created for facile implementation in the clinical setting. More specifically, nucleic acid aptamers for S. moorei were obtained through whole-cell SELEX, and the affinity and specificity of these aptamers were validated. Subsequently, two types of electrochemical sensors were developed. Firstly, an electrochemical impedance spectroscopy sensor was developed to detect impedance changes on the electrode surface, which were caused by the binding of S. moorei to the aptamer. Secondly, a CRISPR/Cas12a-based electrochemical aptasensor was developed based on the ability of the Cas12a enzyme to be activated by specific single-stranded DNA, triggering its trans-cleavage activity. The limits of detection of these sensors for S. moorei were 30 and 6 CFU/mL, respectively. Clinical validation was performed using patient samples to assess the sensor efficacy in a real-world setting. The obtained results suggested that the abundance of S. moorei in the feces of CRC patients was significantly greater compared to that of healthy individuals. This integration of S. moorei aptamers into electrochemical sensors offers a non-invasive and cost-effective alternative to current screening methods available for CRC.
研究领域:
分子诊断学
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生物传感技术
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肿瘤学
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ANALYTICA CHIMICA ACTA
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2026
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IF 6.1
Songbai Tian,Xin Tang,Saijun Zhang,Yuexin Yao,Ran Bi,Haixia Li,Xiaoming Sun,Zhuo Chen
Digital immunoassay allows for the detection of proteins at fg/mL levels by leveraging the principles of Poisson distribution. Nevertheless, the inherent Poisson noise restricts digital sensitivity to targets exceeding 100 molecules, rendering standard digital assays incapable of detecting targets below this threshold. To overcome this constraint, we introduce an integrated CRISPR/Cas12a-digital immunoassay technology. In this system, the CRISPR/Cas12a machinery first acts as a molecular amplifier, converting each target protein into multiple enzymatic reporters. These enzyme molecules are then individually quantified via a digitized readout system, enabling ultrasensitive protein detection. By harnessing the catalytic amplification of CRISPR/Cas12a, the method achieves detection of proteins at copy numbers below 100, effectively surpassing the conventional sensitivity barrier of digital immunoassays. We validated this approach through highly selective and accurate detection of CD44 protein. A linear response was observed across a concentration range of 0.05 to 5 fg/mL, conforming to the calibration model: P(X > 0) = 0.1191c + 0.0036. The limit of detection was determined to be 0.018 fg/mL, equivalent to approximately 36 molecules of CD44. The method was further applied to quantify CD44 in plasma samples from colorectal cancer patients, demonstrating its strong potential for clinical use in early cancer diagnosis and treatment monitoring.
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TALANTA
·
2026
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IF 6.7
Luqin Lv,Yizhuo Zhang,Yaxin Fan,Beining Guo,Yijian Chen
Therapeutic drug monitoring (TDM) of vancomycin (VAN) is critical for maximizing efficacy and minimizing toxicity, but conventional methods are constrained by high costs, slow turnaround times, and operational complexity. To address these limitations, we developed a novel Bivalent Aptamer-assisted CRISPR-Cas12a Sensor (termed BACS) for rapid and precise VAN detection. Central to this platform is a high-affinity bivalent aptamer (2AP33), engineered via molecular docking-guided truncation and rational linker design, which exhibits significantly enhanced binding avidity compared to its monovalent counterpart. This aptamer was integrated into a CRISPR-Cas12a system based on a competitive binding mechanism, where target binding modulates Cas12a trans-cleavage activity. The optimized BACS achieved a wide linear detection range (1-50 μM) with a low limit of detection (0.64 μM) in clinical serum, fully covering the clinical therapeutic window. Notably, the assay is rapid (within 10 min), cost-effective, and simple. Critically, the clinical practicality and reliability of BACS were rigorously validated with 175 clinical serum samples, showing exceptional concordance with both the gold standard method and a classical method. This work not only provides a reliable tool for VAN TDM but also offers an adaptable strategy for developing high-performance CRISPR-powered biosensors for diverse clinical analytes through a streamlined molecular engineering pipeline.
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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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TRENDS IN BIOTECHNOLOGY
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2025
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IF 14.9
Tian Yu,Siyi Zou,Yingxi Long,Yourong Ou,Shixian Liu,Tongjia Kang,Libing Song,Congcong Sun,Guozhen Liu
Developing a generic sensitive platform for detecting diverse biomarkers is essential for a comprehensive understanding of disease states, guiding precision medicine. Herein, we introduce a versatile platform based on glass fiber interfaced CRISPR/Cas with a universal reagent setting (g-CURS), which used a fixed pair of CRISPR RNA (crRNA) and a single-stranded DNA (ssDNA) activator to enable detection of multiple nucleic acids or proteins with ultrahigh sensitivity. The fixed ssDNA activator was labeled on multiple specific ligation products or detection antibodies conjugated on glass fiber to initiate CRISPR/Cas12a-assisted rapid and exponential cascade amplification through circular reporters (CRs), generating fluorescence signals readable by a portable detector. g-CURS was able to detect viral nucleic acids with attomolar sensitivity within 30 min and multiple low-abundance proteins in extracellular vesicles of Parkinson’s disease (PD) serum with subpicomolar sensitivity within 80 min. g-CURS simplifies CRISPR/Cas biosensing using a standard reagent setting, holding promise for biomarker discovery free from bulky instruments.
研究领域:
分子生物学
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医学诊断
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生物传感技术
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SENSORS AND ACTUATORS B-CHEMICAL
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2021
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IF 7.46
Yaqin Zhang,Minyan Chen,Chengrong Liu,Jiaqi Chen,Xinyi Luo,Yingying Xue,Qiming Liang,Li Zhou,Yu Tao,Mingqiang Li,Di Wang,Jianhua Zhou,Jiasi Wang
The outbreak of corona virus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a global pandemic. The high infectivity of SARS-CoV-2 highlights the need for sensitive, rapid and on-site diagnostic assays of SARS-CoV-2 with high-throughput testing capability for large-scale population screening. The current detection methods in clinical application need to operate in centralized labs. Though some on-site detection methods have been developed, few tests could be performed for high-throughput analysis. We here developed a gold nanoparticle-based visual assay that combines with CRISPR/Cas12a-assisted RT-LAMP, which is called C as12a-assisted RT- LA MP/AuN P (CLAP) assay for rapid and sensitive detection of SARS-CoV-2. In optimal condition, we could detect down to 4 copies/μL of SARS-CoV-2 RNA in 40 min. by naked eye. The sequence-specific recognition character of CRISPR/Cas12a enables CLAP a superior specificity. More importantly, the CLAP is easy for operation that can be extended to high-throughput test by using a common microplate reader. The CLAP assay holds a great potential to be applied in airports, railway stations, or low-resource settings for screening of suspected people. To the best of our knowledge, this is the first AuNP-based colorimetric assay coupled with Cas12 and RT-LAMP for on-site diagnosis of COVID-19. We expect CLAP assay will improve the current COVID-19 screening efforts, and make contribution for control and mitigation of the pandemic.
研究领域:
分子生物学
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病毒学
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诊断检测
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纳米技术
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公共卫生
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ACS Sensors
·
2025
·
IF 9.1
Yapeng Wu,Bei Lv,Xintian Ni,Sheng Zhu,Dawei Li
Strand displacement amplification (SDA) is an isothermal DNA amplification technique. Herein, we developed a novel SDA system, designated All-U-Want SDA (AUW-SDA), which was used as a signal amplification strategy for the construction of nucleic acid detection biosensors. AUW-SDA is capable of target turnover and can be utilized for detection of nucleic acid sequences without available 3′-ends. Of particular significance is the ability of AUW-SDA to generate a substantial number of programmable sequences in accordance with the specifications of the sensor signal output methods, irrespective of the sequence of the target nucleic acid. We used the N gene of SARS-CoV-2 as a model target to develop a sensing platform with dual signal outputs. The colorimetric signals were generated by the G-quadruplex/hemin DNAzyme, in which the G-rich sequences were produced by AUW-SDA with a C-rich primer. On the other hand, by altering the sequence within the replaceable region of the primer, an activator sequence was obtained from AUW-SDA, which could trigger the activity of CRISPR/Cas12a, cleaving the probes modified with a fluorophore and quencher at each end and subsequently yielding the fluorescent signals. After the DNA sequences and reaction conditions were optimized, the limit of detection (LOD) values of the fluorescent and colorimetric assays were estimated to be 0.672 fM and 13.3 fM, respectively. The biosensors were utilized for biological sample detection. The reliability of the proposed method was validated against RT-qPCR results. In addition, a portable scanner-assisted high-throughput RGB analysis (PSHRA) method was developed. This method was applied to our biosensor for multilocus detection of SARS-CoV-2. The results obtained were satisfactory, indicating the potential of this approach for field testing or point-of-care (POC) diagnostics.
研究领域:
生物化学
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分子生物学
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诊断学
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ACS Applied Materials & Interfaces
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2024
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IF 8.3
Runlin Yang,Siying Xie,Bin Zhou,Mingming Guo,Jun Fan,Fengli Su,Zhirun Ji,Yue Chen,Bingzhi Li
CRISPR/Cas12a-based biosensing is advancing rapidly; however, achieving sensitive and cost-effective reporting of Cas12a activation remains a challenge. In response, we have developed a label-free system capable of postamplifying Cas12a activation by integrating hybridization chain reaction (HCR) and DNA-copper nanoclusters (DNA-CuNCs). The trans-cleavage of Cas12a triggers a silenced HCR, leading to the in situ assembly of fluorescent DNA-CuNCs, allowing for the turn-on reporting of Cas12a activation. Without preamplification, this assay can detect DNA with a detection limit of 5 fM. Furthermore, when coupled with preamplification, the system achieves exceptional sensitivity, detecting the monkeypox virus (MPXV) plasmid at 1 copy in human serum. In a MPXV pseudovirus-based validation test, the obtained results are in agreement with those obtained by qPCR, reinforcing the robustness of this method. Our study represents the first effort to manipulate DNA-CuNC formation on HCR for highly sensitive and cost-effective reporting of Cas12a, resulting in an efficient synthetic biology-enabled sensing platform for biosafety applications.
研究领域:
合成生物学
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生物传感
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分子诊断
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ANALYTICAL AND BIOANALYTICAL CHEMISTRY
·
2024
·
IF 3.8
Qiao Yinuo,Wang Xiaoyan,Fan Zhenlin,Song Yuzhu,Zhang Jinyang,Han Qinqin
Tuberculosis is a highly infectious disease caused by the bacterium Mycobacterium tuberculosis , and the spread of this agent has caused serious health problems worldwide. The rapid and accurate detection of M. tuberculosis is essential for controlling the spread of infection and for preventing the emergence of multidrug-resistant strains. In this study, the powerful trans-cleavage ability of CRISPR-Cas12a for ssDNA was combined with a surface-enhanced Raman spectroscopy (SERS)–based strategy to establish a CRISPR-SERS sensor for the hypersensitive detection of M. tuberculosis DNA. We observed a linear relationship between the concentration of M. tuberculosis DNA and the output signal over the range of 5 to 100 pM. The equation describing the standard curve was y = 24.10 x + 1594, with R 2 = 0.9914. The limit of detection was as low as 4.42 pM for genomic DNA, and a plasmid containing an M. tuberculosis –specific sequence was detected at 5 copy/μL. A detection accuracy of 100% was achieved in the analysis of DNA isolated from the sputum of hospitalized patients with tuberculosis. The entire detection process is simple to deploy and only takes 50 min and results in the sensitive and specific detection of M. tuberculosis DNA. This study provides a new method for the detection of tuberculosis. The tool is stable and can be utilized on-site, and it thus broadens the diagnostic application of CRISPR-Cas12a-based sensor technology.
研究领域:
医学诊断
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分子生物学
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