Bioengineered
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2022
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IF 6.832
Wei-Hao Wu,Xiao-Mei Ma,Jian-Qing Huang,Qin Lai,Fu-Neng Jiang,Cui-Yun Zou,Long-Tian Chen,Lian Yu
This study investigated whether CRISPR/Cas9 (D10A) nickase-mediated gene editing can correct the aberrant Hb Constant Spring mutation (Hb CS or HBA2: c.427 T > C) in fibroblasts. Vectors for repairing the α-globin-encoding gene, HBA2:c.427 T > C mutation, includingthe CRISPR/Cas9(D10A)-sg plasmid and donor with homology arms, were constructed and used to perform gene editing in patient-derived fibroblasts. We subsequently analyzed the genetic correction, the gene editing efficiency and off-target effect. Sequencing analysis and the BamHI assay showed that HB CS mutant cells were repaired with Hb CS point mutations, the editing efficiency was 4.18%~9.34% and no off-target effects were detected. The results indicate that the HB CS mutant gene is effectively repaired by the CRISPR/Cas9 (D10A)system, which may enable truly personalized therapy for precise repair of α-thalassemia.
研究领域:
分子生物学
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遗传学
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医学
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DOI
PubMed
高影响因子
TALANTA
·
2025
·
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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DOI
PubMed
高影响因子
Nature Communications
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2025
·Vol. 16
·期 1
·1-14
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IF 15.7
Hu Hao,Guo Songcheng,Li Yiyuan,Dong Kejun,Lu Yan,Ye Keyi,Li Longjie,Zhou Xiaoyu,Cheng Liming,Xiao Xianjin
Detecting small molecules is pivotal across fields like clinical diagnostics, environmental monitoring, and food safety. The CRISPR-Cas12a system, known for its simplicity and sensitivity, offers a promising basis for small molecule detection. However, current CRISPR-based detection methods face challenges, including complex design requirements, high background noise, and limited adaptability to different targets. In our study, we introduce the SBS-Cas system, leveraging a split crRNA mode to induce spatial hindrance on the scaffold strand through molecular binding. This approach prevents the assembly with Cas12a, effectively masking its trans-cleavage activity. By introducing small molecules that competitively bind to the macromolecule, we eliminate this spatial hindrance, activating Cas12a. Our results demonstrate high sensitivity, versatility, and adaptability in small molecule detection across multiple reactions, with successful intracellular imaging and responsive fluctuations in complex environments underscoring the system’s robustness. This innovative CRISPR-Cas12a-based approach establishes a low-background, highly sensitive platform for small molecule detection. SBS-Cas promises not only to enhance tools for clinical, environmental, and food safety applications but also to advance CRISPR research, providing insights and expanding possibilities in molecular detection science.
研究领域:
分子生物学
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临床诊断
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环境科学
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食品安全
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DOI
PubMed
高影响因子
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.
研究领域:
分子生物学
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医学检测
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生物技术
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DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2024
·
IF 5.7
Jihua Wei,Jiayi Zhang,Wei Wang,Haidong Zhou,Huade Ma,Yuanxun Gong,Qianli Tang,Kai Zhang,Xianjiu Liao
The article details a groundbreaking platform for detecting microRNAs (miRNAs), crucial biomolecules involved in gene regulation and linked to various diseases. This innovative platform combines the CRISPR-Cas13a system's precise ability to specifically target and cleave RNA molecules with the amplification capabilities of the hybridization chain reaction (HCR). HCR aids in signal enhancement by creating branched DNA structures. Additionally, the platform employs electrochemiluminescence (ECL) for detection, noted for its high sensitivity and low background noise, making it particularly effective. A key application of this technology is in the detection of miR-17, a biomarker associated with multiple cancer types. It exhibits remarkable detection capabilities, characterized by low detection limits (14.38 aM) and high specificity. Furthermore, the platform's ability to distinguish between similar miRNA sequences and accurately quantify miR-17 in cell lysates underscores its significant potential in clinical and biomedical fields. This combination of precise targeting, signal amplification, and sensitive detection positions the platform as a powerful tool for miRNA analysis in medical diagnostics and research.
研究领域:
生物技术
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分子生物学
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医学诊断
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癌症研究
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DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2021
·
IF 6.986
Wei S. Zhang,Jianbin Pan,Feng Li,Min Zhu,Mengting Xu,Hongyan Zhu,Yanyan Yu,Gaoxing Su
The outbreak of the pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) calls for an urgent unmet need for developing a facial and cost-effective detection method. The requirement of well-trained personnel and sophisticated instrument of current primary mean (reverse transcription polymerase chain reaction, RT-PCR) may hinder the practical application worldwide. In this regard, a reverse transcription recombinase polymerase amplification (RT-RPA) coupled with CRISPR-Cas12a colorimetric assay is proposed for the SARS-CoV-2 detection. The methodology we have described herein utilizes DNA-modified gold nanoparticles (AuNPs) as a universal colorimetric readout and can specifically target ORF1ab and N regions of the SARS-CoV-2 genome. After the virus genome is amplified through RT-RPA, the resulting abundant dsDNA will bind and activate Cas12a. Under trans-cleavage degradation, the capped DNA substrate will be hydrolyzed gradually from AuNPs, demonstrating a change in the surface plasmon resonance (SPR), which can be facially monitored by UV–vis absorbance spectroscopy and naked eye observation. The high amplification efficiency from RT-RPA and Cas12a trans-cleavage process bring the sensitivity of our method to 1 copy of viral genome sequence per test. Notably, under the dual variations inspecting from the isothermal amplification and Cas12a activation process, the false positive events from other beta coronavirus members can be effectively avoided and thus significantly improve the specificity. Furthermore, the reliability of this colorimetric assay is validated by standard clinical samples from the hospital laboratory department. Through integration of the inherently high sensitivity and specificity from an RPA-coupled Cas12a system with the intrinsic simplicity of AuNP-based colorimetric assay, our method increases the practical testing availability of SARS-CoV-2.
研究领域:
分子生物学
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病毒学
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诊断学
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DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2021
·
IF 6.558
Yanju Chen,Hui Wu,Siwenjie Qian,Xiaoping Yu,Huan Chen,Jian Wu
Heavy metal lead accumulation in the environment pollutes the ecology systems and further threatens the human health. It is necessary to develop a sensitive method to detect it. Here, we propose a highly sensitive lead detection method by combining DNAzyme and CRISPR system. Once the lead ion is recognized, the substrate chain of DNAzyme is cleaved to produce single strand DNA . The produced single strand DNA can be detected by Cas protein/guide RNA complex and further trigger the collateral cleavage effect of CRISPR system, which can indiscriminately cut short single strand DNA reporters. By this way, the detection signals can be greatly amplified. This method can detect lead ions as low as 0.48 nM. The sensitivity is higher than the DNAzyme method. Furthermore, the portable 3D printing device is designed to observe the fluorescent signals so the end-point detection results can be visualized by the naked eyes. The entire detection process can avoid using bulky and expensive instruments, which can promote on-site lead ion detection.
研究领域:
生物化学
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分子生物学
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环境科学
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DOI
PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
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2021
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IF 7.46
Lijuan Yin,Ninghui Duan,Si Chen,Yuan Yao,Jifeng Liu,Long Ma
Foodborne diseases, caused by pathogenic bacteria, severely threaten global human health and cause a financial burden. Rapid, sensitive and on-site detection of pathogenic bacteria is significant. The existing methods have different defects, such as time-consuming and inconvenient. In this study, we developed a G-quadruplex-based CRISPR-Cas12a bioassay for pathogenic bacteria detection with high sensitivity and visualization capability. Salmonella was used as the detection model. Simply, the amplicons of Salmonella specific invA gene activated the trans -cleavage activity of Cas12a and triggered CRISPR-Cas12a based indiscriminate degradation of single-stranded DNAs (ssDNAs). The ssDNAs were designed with the guanine-rich sequence and formed a stable G-quadruplex DNAzyme by adding K + . This DNAzyme could catalyze the TMB-H 2 O 2 reaction in the presence of hemin, leading to an increase in absorbance at 454 nm and a color change. This change can be readily differentiated by the naked eyes as well as a smartphone with a Color Picker App. With this strategy, the limit of detection (LOD) for Salmonella was 1 CFU/mL with no cross-reactivity. A linear relationship (R 2 = 0.993) between the absorbance and the concentration of Salmonella was obtained. Furthermore, G-quadruplex-based CRISPR-Cas12a bioassay was successfully applied for sensing Salmonella in real food samples. This work not only expands the reach of CRISPR-Cas based biosensing but also provides a novel pathogenic bacteria detection method with high sensitivity, specificity and on-site capability.
研究领域:
食品科学
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分子生物学
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生物技术
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DOI
高影响因子
TALANTA
·
2022
·
IF 6.556
Yaqin Zhang,Xiangyu Quan,Yingchun Li,Hangyu Guo,Fange Kong,Jiahui Lu,Lirong Teng,Jiasi Wang,Di Wang
The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic, highlighting the unprecedented demand for rapid and portable diagnostic methods. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) proteins-based platforms have been used for the detection of pathogens. However, in further applications and research, due to multiple steps needed, many methods showed an increased risk of cross-reactivity. The thermostable Cas12b enables the combination of isothermal amplification and CRISPR-mediated detection, which could decrease the risk of cross-contamination. In this study, we developed a portable and specific diagnostic method that combined the gold nanoparticle (AuNP) with thermal stable CRISPR/Cas12b-enhanced reverse transcription loop-mediated isothermal amplification (RT-LAMP), which is called SCAN, to distinguish the N gene of SARS-CoV-2 from flu gene. We validated our method using RNA from cells transfected by plasmids. We could easily distinguish the positive results by the naked eye based on the strong molar absorption coefficient of AuNP. Moreover, SCAN has the potential for high-throughput tests owing to its convenient operation. In sum, SCAN has broken the site and equipment restrictions of traditional detection methods and could be applied outside of hospitals and clinical laboratories, greatly expanding the test of COVID-19.
研究领域:
分子生物学
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生物技术
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病毒学
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诊断医学
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产品关联
DOI
高影响因子
MICROCHEMICAL JOURNAL
·
2025
·
IF 5.1
Ping Qiu,Longjian Huang,Jiayi Zhang,Wenbo Li,Xudong Xin,Luchang Chen,Qianli Tang,Kai Zhang,Xianjiu Liao,Jihua Wei
We report the development of a novel split aptamer-driven electrochemical biosensor integrating entropy-driven Signal amplification (EDSA) and CRISPR-Cas12a technology for the highly sensitive and specific detection of adenosine deaminase (ADA). ADA is an important biomarker involved in immune regulation and purine metabolism, and its accurate quantification is critical for diagnosing various diseases. In our system, ADA catalyzes the deamination of adenosine to inosine, resulting in the release of Aptamer1 from a split aptamer structure. The liberated Aptamer1 initiates a strand displacement reaction within a Blocker/Bandage/Scaffold complex, triggering a cascade of entropy-driven amplification cycles that generate a DNA duplex capable of activating CRISPR-Cas12a. Activated Cas12a exhibits trans -cleavage activity, specifically cleaving a ferrocene-labeled single-stranded DNA (ssDNA) probe immobilized on a gold electrode, leading to a measurable decrease in electrochemical signal. The biosensor operates under isothermal and enzyme-free conditions, providing simplicity, rapid detection, and low cost. It achieves a low detection limit of 0.688 U·L −1 with a wide linear range from 1 to 20,000 U·L −1 . The system demonstrates excellent specificity against ADA analogues and common interfering proteins and performs reliably in diluted human serum samples. This strategy represents a versatile and modular platform for point-of-care diagnostics. By altering the aptamer sequence, the biosensor can be adapted for the detection of other clinically relevant proteins and enzymes, contributing to advances in personalized medicine.
研究领域:
生物传感技术
·
分子诊断
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医学检测
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DOI
高影响因子
ACS Synthetic Biology
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2022
·
IF 5.249
Ming Zhao,Miaomiao Gao,Liangbin Xiong,Yongjun Liu,Xinyi Tao,Bei Gao,Min Liu,Feng-Qing Wang,Dong-Zhi Wei
Genome mutagenesis drives the evolution of organisms. Here, we developed a CRISPR-Cas assisted random mutation (CARM) technique for whole-genome mutagenesis. The method leverages an entirely random gRNA library and SpCas9-NG to randomly damage genomes in a controllable shotgunlike manner that then triggers diverse and abundant mutations via low-fidelity repair. As a proof of principle, CARM was applied to evolve the capacity of Saccharomyces cerevisiae BY4741 to produce β-carotene. After seven rounds of iterative evolution over two months, a β-carotene hyperproducing strain, C7-143, was isolated with a 10.5-fold increase in β-carotene production and 857 diverse genomic mutations that comprised indels, duplications, inversions, and chromosomal rearrangements. Transcriptomic analysis revealed that the expression of 2541 genes of strain C7-143 was significantly altered, suggesting that the metabolic landscape of the strain was deeply reconstructed. In addition, CARM was applied to evolve industrially relevant S. cerevisiae CEN.PK2-1C for S-adenosyl-L-methionine production, which was increased 2.28 times after just one round. Thus, CARM can contribute to increasing genetic diversity to identify new phenotypes that could further be investigated by reverse engineering.
研究领域:
遗传学
·
合成生物学
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微生物学
·
代谢工程
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产品关联
DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2023
·
IF 6.2
Shiqi Xu,Songtao Wang,Ling Guo,Yuqin Tong,Lina Wu,Xingxu Huang
CRISPR-based detection was often based on the target preamplification to realize the high sensitivity. Here, we prepared a CRISPR-Cas12a system for the colorimetric detection of lead ion (Pb 2+ ) based on the assistance of DNAzyme and nanozyme instead of preamplification. The recognition between GR-5 DNAzyme and Pb 2+ could trigger the CRISPR-Cas12a system. MnO 2 nanozymes connected with magnetic beads through single stranded DNA were prepared as the colorimetric signal probes and catalyst of CRISPR-Cas12a system for the strong oxidase-like activity inducing the color change of 3,3′,5,5′-tetramethylbenzidine. The nanozyme-catalysed CRISPR-Cas12a system could be used to detect Pb 2+ through the color change with high specificity and sensitivity. The linear range of this approach was 0.8 nM–2500 nM, with a limit of detection of 0.54 nM. This method was applied for the detection of the Pb 2+ in food samples indicating good accuracy and anti-interference ability.
研究领域:
生物传感器
·
分析化学
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环境科学
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食品安全
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产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）