Cell Host & Microbe
·
2023
·
IF 30.3
Chao Liu,Rui Wang,Jie Li,Feiyue Cheng,Xian Shu,Huiwei Zhao,Qiong Xue,Haiying Yu,Aici Wu,Lingyun Wang,Sushu Hu,Yihan Zhang,Jun Yang,Hua Xiang,Ming Li
CRISPR RNAs (crRNAs) and Cas proteins work together to provide prokaryotes with adaptive immunity against genetic invaders like bacteriophages and plasmids. However, the coordination of crRNA production and cas expression remains poorly understood. Here, we demonstrate that widespread modulatory mini-CRISPRs encode cas-regulating RNAs (CreRs) that mediate autorepression of type I-B, I-E, and V-A Cas proteins, based on their limited complementarity to cas promoters. This autorepression not only reduces autoimmune risks but also responds to changes in the abundance of canonical crRNAs that compete with CreR for Cas proteins. Furthermore, the CreR-guided autorepression of Cas proteins can be alleviated or even subverted by diverse bacteriophage anti-CRISPR (Acr) proteins that inhibit Cas effectors, which, in turn, promotes the generation of new Cas proteins. Our findings reveal a general RNA-guided autorepression paradigm for diverse Cas effectors, shedding light on the intricate self-coordination of CRISPR-Cas and its transcriptional counterstrategy against Acr proteins.
研究领域:
分子生物学
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微生物学
·
遗传学
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DOI
PubMed
高影响因子
Research
·
2022
·
IF 11.036
Liangfen Cheng,Fuhan Yang,Longfei Tang,Lelin Qian,Xu Chen,Feng Guan,Juan Zhang,Genxi Li
Evaluating tumor development is of great importance for clinic treatment and therapy. It has been known that the amounts of sialic acids on tumor cell membrane surface are closely associated with the degree of cancerization of the cell. So, in this work, cellular interface supported CRISPR/Cas trans-cleavage has been explored for electrochemical simultaneous detection of two types of sialic acids, i.e., N-glycolylneuraminic acid (Neu5Gc) and N-acetylneuraminic acid (Neu5Ac). Specifically, PbS quantum dot-labeled DNA modified by Neu5Gc antibody is prepared to specifically recognize Neu5Gc on the cell surface, followed by the binding of Neu5Ac through our fabricated CdS quantum dot-labeled DNA modified by Sambucus nigra agglutinin. Subsequently, the activated Cas12a indiscriminately cleaves DNA, resulting in the release of PbS and CdS quantum dots, both of which can be simultaneously detected by anodic stripping voltammetry. Consequently, Neu5Gc and Neu5Ac on cell surface can be quantitatively analyzed with the lowest detection limits of 1.12 cells/mL and 1.25 cells/mL, respectively. Therefore, a ratiometric electrochemical method can be constructed for kinetic study of the expression and hydrolysis of Neu5Gc and Neu5Ac on cell surface, which can be further used as a tool to identify bladder cancer cells at different development stages. Our method to evaluate tumor development is simple and easy to be operated, so it can be potentially applied for the detection of tumor occurrence and development in the future.
研究领域:
生物医学工程
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分子生物学
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肿瘤学
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DOI
高影响因子
Journal of the Taiwan Institute of Chemical Engineers
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2015
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IF 3
Changxin Li,Hong Zhong,Shuai Wang,Jianrong Xue,Haibo Wang
In this study, using coal as a reducer, the thermal decomposition of CaSO 3 to produce CaS was carried out at different conditions. The mineralogical composition and microstructure of the as-obtained CaS were analyzed by scanning electron microscopy (SEM) and X-ray diffraction (XRD). Results revealed that single-phase CaS was detected in the product under optimum conditions and the optimal synthesis conditions of CaS were as follows: molar ratio of C and CaSO 3 (C/S ratio)=2.5, reaction temperature 1173 K, reaction time 1.5 h and coal particle size 100 mesh. Under the optimum conditions, the purity and yield of CaS are up to 99.15% and 96.52% respectively, which indicated that the decomposition was almost complete and the as-synthesized CaS can be recycled to be a raw material in chemical industry. Meanwhile, thermodynamic calculation showed that the reaction for CaS generation was very complex and CaS was generated mainly through solid–solid reaction and gas–solid reaction.
研究领域:
材料科学
·
化学工程
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DOI
高影响因子
TRANSACTIONS OF NONFERROUS METALS SOCIETY OF CHINA
·
2015
·
IF 1.178
Chang-xin LI,Hong ZHONG,Shuai WANG,Jian-rong XUE,Fang-fang WU,Zhen-yu ZHANG
The extraction of manganese from low-grade manganese oxide ores using CaS derived from CaSO 4 as reductant was investigated. The effects of mass ratio of CaS to ore, reduction temperature, reduction time, liquid to solid ratio (L/S ratio), stirring speed, leaching temperature, leaching time and H 2 SO 4 concentration on the leaching rates of Mn and Fe during the reduction–acid leaching process were discussed. The leaching rates of 96.47% for Mn and 19.24% for Fe were obtained under the optimized conditions of mass ratio of CaS to manganese oxide ore 1:6.7, L/S ratio 5:1, stirring speed 300 r/min, reduction temperature of 95 °C for 2.0 h in the reduction process and leaching stirring speed of 200 r/min, H 2 SO 4 concentration of 1.5 mol/L, leaching temperature of 80 °C for 5 min in the leaching process. In addition, this process can be employed in the recovery of manganese from various manganese oxide ores, and Mn leaching rate above 95% is obtained.
研究领域:
冶金工程
·
化学工程
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产品关联
DOI
高影响因子
Nature Protocols
·
2014
·Vol. 9
·期 10
·2493-2512
·
IF 7.782
Shao Yanjiao,Guan Yuting,Wang Liren,Qiu Zhongwei,Liu Meizhen,Chen Yuting,Wu Lijuan,Li Yongmei,Ma Xueyun,Liu Mingyao,Li Dali
Conventional embryonic stem cell (ESC)–based gene targeting, zinc-finger nuclease (ZFN) and transcription activator–like effector nuclease (TALEN) technologies are powerful strategies for the generation of genetically modified animals. Recently, the CRISPR/Cas system has emerged as an efficient and convenient alternative to these approaches. We have used the CRISPR/Cas system to generate rat strains that carry mutations in multiple genes through direct injection of RNAs into one-cell embryos, demonstrating the high efficiency of Cas9-mediated gene editing in rats for simultaneous generation of compound gene mutant models. Here we describe a stepwise procedure for the generation of knockout and knock-in rats. This protocol provides guidelines for the selection of genomic targets, synthesis of guide RNAs, design and construction of homologous recombination (HR) template vectors, embryo microinjection, and detection of mutations and insertions in founders or their progeny. The procedure from target design to identification of founders can take as little as 6 weeks, of which <10 d is actual hands-on working time.
研究领域:
分子生物学
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遗传学
·
实验动物学
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产品关联
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
ACS Synthetic Biology
·
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
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代谢工程
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产品关联
DOI
PubMed
高影响因子
ACS Sensors
·
2025
·
IF 9.1
Shixian Liu,Tian Yu,Libing Song,Kourosh Kalantar-Zadeh,Guozhen Liu
Highly sensitive point-of-care early screening for high-risk human papillomavirus (HPV) infections is urgently needed, particularly in resource-limited settings. Nucleic acid amplification methods, especially CRISPR/Cas-based biosensors, have emerged as promising tools for sensitive HPV detection; however, current approaches typically rely on tedious tube-based formats coupled with lateral flow assays for signal readout in point-of-care testing (POCT). Here, we developed customized microfluidic paper-based analytical devices (μPADs) with valves that seamlessly integrated recombinase polymerase amplification (RPA) with CRISPR/Cas12a biosensing (RPA-CRISPR/Cas12a) on the filter paper substrate. This innovation achieved sensitive and cost-effective high-risk HPV detection in POCT. The RPA-CRISPR/Cas12a system with a linear reporter on μPADs, enabled fluorescence detection of the E7 gene, achieving a sensitivity of 1 pM at approximately 1 h. The sensitivity was further enhanced by introducing a circular reporter into the fluorescence-based RPA-CRISPR/Cas12a system on μPADs, enabling detection of the E7 gene with a detection limit of 1 fM and an assay time of 35 min. The system was validated using 50 cervical swab clinical samples, demonstrating 95% sensitivity and 100% specificity when compared to qPCR. This sample-to-answer detection platform holds significant promise for early screening of high-risk HPV infections in point-of-care scenarios.
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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产品关联
DOI
PubMed
高影响因子
Theranostics
·
2025
·Vol. 15
·期 7
·2870
·
IF 13.3
Xuena Zhu,Shanshan Wang,Yuanyuan Xue,Xiaoyan Wang,Shaoqi Hu,Tingbo Liang,Wenjun Liu
Significant efforts and resources have been dedicated to developing CRISPR-Cas based point-of-care testing (POCT) and self-diagnosis methods for nucleic acid pathogens, aiming to complement the gold standard quantitative PCR tests, particularly in settings where centralized facilities, trained personnel, or resource-intensive equipment are unavailable. However, the reliance on stationary, high-cost readout machinery hinders their full deployment at the point of care. We aimed to develop a solid-phase invertase-labeled reporter (ILR) system that enables convenient readout of CRISPR-Cas reactions, facilitate HPV detection in a POCT-compatible manner. Methods: Through multiple chemical couplings, invertase is immobilized onto magnetic microbeads via a nucleic acid linker that responds to target nucleic acid-induced CRISPR-Cas activation. This activation releases active invertase, which then converts sucrose to glucose in proportion to the target's abundance. Enzymatic signal amplification by Cas12a/Cas13a and invertase compensates for the moderate sensitivity of personal glucose meters (PGMs). Results: When applied to human papillomavirus detection, the HPV18-targeted LAMP-Cas12a/ILR/PGM system can detect as few as 7 HPV18-positive HeLa cells out of 7,000, achieving 95.8% sensitivity and 100% specificity in cervical cell samples. Furthermore, minimal reagent adjustments allow for the rapid establishment of HPV16 and HPV52-targeted LAMP-Cas12a/ILR/PGM systems, offering satisfactory sensitivity, specificity, and cross-species detection. Conclusion: These findings demonstrate a highly efficient testing platform for a range of nucleic acid pathogens, suitable for both point-of-care and household use.
研究领域:
分子生物学
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医学诊断
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生物化学
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产品关联
DOI
PubMed
高影响因子
ACS Applied Materials & Interfaces
·
2024
·
IF 8.3
Wenjing Lei,Liangwen Hao,Han Qiu,Kexin Bian,Tianming Cui,Weiwei Zeng,Yu Zhang,Weitao Yang,Bingbo Zhang
Nucleic acid detection plays a pivotal role in the accurate diagnosis of diseases. The CRISPR/Cas detection system, noted for its significant utility in a variety of applications, often necessitates enhanced sensitivity or specific signal amplification strategies, particularly for detecting low-abundance biomarkers. In this study, we present a quantum-dot-encoded beads (QDB)-energized CRISPR/Cas12-based lateral-flow assay (QDB-CRISPR-LFA). This method enables amplification-free, sensitive, and rapid detection (<40 min) of BRCA-1. We validated our method using contrived reference samples and nucleic acids extracted from tumor cells. The QDB-CRISPR-LFA provides a visual, more rapid alternative to the traditional BRCA-1 real-time RT-PCR assay. Significantly, through the integration of CRISPR’s specificity and the high signal output of QDB, the detection threshold for BRCA-1 has been reduced to the femtomolar level, representing an enhancement of 2–4 orders of magnitude over existing CRISPR/Cas detection methods. This advancement underscores the potential of our approach in advancing nucleic acid detection techniques, which is crucial for the early and precise diagnosis of diseases.
研究领域:
分子生物学
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生物技术
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医学诊断
·
遗传学
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产品关联
DOI
PubMed
高影响因子
JOURNAL OF HAZARDOUS MATERIALS
·
2023
·
IF 13.6
Long Ma,Wenlu Zhang,Lijuan Yin,Yaru Li,Jianwen Zhuang,Liang Shen,Shuli Man
The pandemic of COVID-19 creates an imperative need for sensitive and portable detection of SARS-CoV-2. We devised a SERS-read, CRISPR/Cas-powered nanobioassay, termed as OVER-SARS-CoV-2 (One-Vessel Enhanced RNA test on SARS-CoV-2), which enabled supersensitive, ultrafast, accurate and portable detection of SARS-CoV-2 in a single vessel in an amplification-free and anti-interference manner. The SERS nanoprobes were constructed by conjugating gold nanoparticles with Raman reporting molecular and single-stranded DNA (ssDNA) probes, whose aggregation-to-dispersion changes can be finely tuned by target-activated Cas12a though trans -cleavage of linker ssDNA. As such, the nucleic acid signals could be dexterously converted and amplified to SERS signals. By customizing an ingenious vessel, the steps of RNA reverse transcription, Cas12a trans -cleavage and SERS nanoprobes crosslinking can be integrated into a single and disposal vessel. It was proved that our proposed nanobioassay was able to detect SARS-CoV-2 as low as 200 copies/mL without any pre-amplification within 45 min. In addition, the proposed nanobioassay was confirmed by clinical swab samples and challenged for SARS-CoV-2 detection in simulated complex environmental and food samples. This work enriches the arsenal of CRISPR-based diagnostics (CRISPR-Dx) and provides a novel and robust platform for SARS-CoV-2 decentralized detection, which can be put into practice in the near future.
研究领域:
分子诊断
·
纳米技术
·
病毒学
·
生物医学工程
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产品关联
DOI
PubMed
高影响因子
Animal Nutrition
·
2026
·
IF 8.3
Yu Wang,Yan Zhang,Liyun Wu,Chaoyue Li,Qiaozhen Chen,Dong Han,Haokun Liu,Zhimin Zhang,Shouqi Xie,Junyan Jin
As a precursor to creatine, guanidinoacetic acid (GAA) is widely recognized to enhance growth performance and flesh quality of animals, but the underlying molecular mechanism remains unclear. This study evaluated the effects of dietary GAA supplementation on growth performance, textural properties, and flavor constituents in gibel carp CAS V ( Carassius gibelio , CAS V). A total of 300 healthy gibel carp (5.01 ± 0.13 g) at 42 d of age were assigned randomly to 12 tanks (3 replicates per group, 25 fish per replicate). Fish were fed with a basal diet supplemented with graded levels of GAA (0%, 0.03%, 0.06%, and 0.12%) for 10 weeks. Results demonstrated that GAA supplementation significantly enhanced the specific growth rate (SGR) and crude protein content in gibel carp ( P < 0.05). It also enhanced muscle physicochemical attributes by increasing water holding capacity and hardness, along with boosting glycogen content and free glutamate level ( P < 0.05). Mechanistically, GAA promoted myofiber development by upregulating protein and gene expression of myogenic regulatory factors (MRFs), leading to increase myofiber density and a higher frequency of myofibers with diameters between 20 and 40 μm ( P < 0.05). Furthermore, GAA facilitated collagen synthesis to improve muscle hardness by activating the transforming growth factor-beta 1 (TGF-β1)/mothers against decapentaplegic homolog (SMADs) signaling pathway, upregulating transcript levels of tgf-β1 , col1α1 , col1α2 , and smad3 ( P < 0.05). Additionally, GAA increased inosine monophosphate (IMP) content in muscle ( P = 0.015), which was associated with enhanced expression of AMPD1 protein and upregulation of the adsl , pkm , and ckm genes ( P < 0.05). In conclusion, dietary GAA supplementation enhanced flesh quality of gibel carp via improving growth performance, nutrient deposition, texture characteristics, and flavor components.
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DOI
PubMed
高影响因子
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