BIOTECHNOLOGY AND BIOENGINEERING
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2021
·Vol. 118
·期 5
·2053-2066
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IF 4.53
Wei Lin,Tian Tian,Yongzhong Jiang,Erhu Xiong,Debin Zhu,Xiaoming Zhou
Polymerase chain reaction (PCR), a central technology for molecular diagnostics, is highly sensitive but susceptible to the risk of false positives caused by aerosol contamination, especially when an end-point detection mode is applied. Here, we proposed a solution by designing a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 eraser strategy for eliminating potential contamination amplification. The CRISPR/Cas9 engineered eraser is firstly adopted into artpcr reverse-transcription PCR (RT-PCR) system to achieve contamination-free RNA detection. Subsequently, we extended this CRISPR/Cas9 eraser to the PCR system. We engineered conventional PCR primers to enable the amplified products to contain an implanted NGG (protospacer adjacent motif, PAM) site, which is used as a code for specific CRISPR/Cas9 recognition. Pre-incubation of Cas9/sgRNA with PCR mix leads to a selective cleavage of contamination amplicons, thus only the template DNA is amplified. The developed CRISPR/Cas9 eraser, adopted by both RT-PCR and PCR systems, showed high-fidelity detection of SARS-CoV-2 and African swine fever virus with a convenient strip test.
研究领域:
分子生物学
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医学诊断
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基因编辑
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产品关联
DOI
PubMed
高影响因子
Fermentation-Basel
·
2024
·Vol. 10
·期 11
·589
·
IF 3.3
Jiaojiao He,Haoyang Zhou,Jine Liang,Kadireya Tuerxun,Zhuoling Ding,Shishui Zhou
In typical whiskey, the content of higher alcohols is about 1500–2000 mg/L, leading to a high intoxicating degree (ID). To produce low-ID whiskey,Saccharomyces cerevisiaeXF0-h, XF0-H and XF0-LH were successfully constructed by CRISPR-Cas9 gene editing technology to knockoutHOM2(encoding aspartate β-semialdehyde dehydrogenase) in the original strain XF0 and theLEU1knockout strain XF0-L. The contents of higher alcohols in whiskey fermented by XF0-h, XF0-H, and XF0-LH were 704 ± 8 mg/L, 685 ± 6 mg/L, and 685 ± 19 mg/L, respectively, showing reductions of 23.93%, 25.98%, and 15.81% compared to XF0, XF0, and XF0-L. The fermentation conditions of XF0-LH were optimized through single-factor experiments and the Box–Behnken design. The optimal conditions were a wort concentration of 9.8 °P, hydrolyzed broken rice syrup addition of 78 g/L, and an inoculum size of 2.7 × 106cells/mL. The low-ID whiskey was brewed with a higher alcohol content of 556 mg/L by 50 L fermenter at the optimal conditions.
研究领域:
生物技术
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发酵工程
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食品科学
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产品关联
DOI
高影响因子
Nano Today
·
2021
·
IF 20.722
Dagan Zhang,Lijun Cai,Xiaowei Wei,Yuetong Wang,Luoran Shang,Lingyun Sun,Yuanjin Zhao
Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated nucleases (CRISPR/Cas) platforms have a demonstrated value in nucleic acid detection, while their multiplex screening capability is still challenging. Herein, we present a novel CRISPR/Cas9 platform based on bioinspired photonic crystal (PhC) barcodes for multiplex and sensitive nucleic acid detection. The bioinspired PhC barcodes not only exhibit distinctive structural color as encoding elements, but also possess abundant functional surface groups for probe immobilization with the presence of polydopamine (PDA) coating. The CRISPR/Cas9 system recognizes and cleaves target DNA and with the help of Klenow fragment to produce ssDNA, which was subsequently captured by the PhC barcodes and the detection signal was amplified by hybridization chain reaction (HCR). Based on this platform, we demonstrated that Human Papilloma Virus (HPV) nucleic acids could be detected with a highly sensitive limit of 0.025 pM and be multiplexed assayed with high accuracy and specificity. Thus, we believe that this platform paves an avenue for multiplexed biomarkers quantification in clinical disease diagnostics.
研究领域:
分子生物学
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诊断学
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生物医学工程
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产品关联
DOI
高影响因子
Cell Reports Medicine
·
2025
·
IF 10.6
Jianhai Chen,Jianwei Tan,Nannan Wang,Hui Li,Wenxiang Cheng,Jian Li,Benguo Wang,Adam C. Sedgwick,Zhitong Chen,Guojun Chen,Peng Zhang,Wei Zheng,Chengbo Liu,Jingqin Chen
Rheumatoid arthritis (RA) is the most prevalent inflammatory arthritis with unknown etiology, characterized by synovial inflammation and articular bone erosion. Studies have highlighted that inhibiting macrophage-induced osteoclastogenesis holds promise in mitigating bone destruction. However, specifically halting this pathological cascade remains a challenge for the management of RA. Here, initially, we identify that Ras homolog gene family member A ( RhoA ) is a pivotal target in inducing osteoclastogenesis of macrophages. Subsequently, we develop a strategy termed specific macrophages RhoA targeting (SMART), in which phosphatidylserine (PS)-enriched macrophage membranes are engineered to deliver macrophage-specific promoter-containing CRISPR-Cas9 plasmids (SMART-Cas9), enabling targeted editing of RhoA in RA joint macrophages. Multiscale imaging techniques confirm the highly specific targeted effect of SMART-Cas9 on the macrophages of inflamed joints. SMART-Cas9 successfully reduces osteoclastogenesis by macrophages, thus mitigating bone erosion by modulating cytoskeletal dynamics and immune balance in inflammatory arthritis, representing a therapeutic avenue for RA and other inflammatory bone diseases.
研究领域:
免疫学
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分子生物学
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风湿病学
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产品关联
DOI
PubMed
高影响因子
Chemical Science
·
2022
·Vol. 13
·期 38
·11433-11441
·
IF 9.969
Xuan Zhao,Na Na,Jin Ouyang
The biological state at the subcellular level is highly relevant to many diseases, and the monitoring of organelles such as mitochondria is crucial based on this. However, most DNA and protein based nanoprobes used for the detection of mitochondrial RNAs (mitomiRs) lack spatial selectivity, which leads to inefficiencies in probe delivery and signal turn-on. Herein, we constructed a novel DNA nanoprobe named protein delivery nano-corona (PDNC) to improve the delivery efficiency of Cas protein, for spatially selective imaging of mitomiRs in living cells switched on by a CRISPR/Cas system. Combined with a single-molecule counting method, this strategy enables highly sensitive detection of low-abundance mitomiR. Therefore, the strategy in this work opens up new opportunities for cell identification, early clinical diagnosis, and research in biological behaviour at the subcellular level.
研究领域:
分子生物学
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纳米技术
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细胞生物学
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基因组学
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产品关联
DOI
PubMed
高影响因子
ACS Nano
·
2025
·
IF 16
Hui Liu,Yanting Yang,Nasha Zhang,Yuebing Hou,Ziqi Zhang,Xinyuan Yu,Yizhou Huang,Bowen Yan,Houren Zhou,Mengyao Mu,Yuan Gao,Kai Guo,Ming Yang,Xiao Sun
The development of new CRISPR/Cas9 delivery systems with a synergistic therapeutic mode can provide a new perspective for efficient tumor treatment. In this work, we developed a bionic 2D FeS nanoplatform with high CRISPR/Cas9 loading (FCRM), highlighting the synergy of CRISPR/Cas9 and ferroptosis in regulating heat shock proteins (HSPs) for enhanced tumor photothermal therapy (PTT). Due to the ultrathin structure and pH-response of FeS nanosheets, FCRM can quickly decompose in a tumor microenvironment and effectively release CRISPR/Cas9 and Fe2+, which can be further enhanced by a photothermal process. CRISPR/Cas9 can accurately downregulate the level of intracellular antiapoptosis protein Survivin. Fe2+ can induce lipid peroxidation and ferroptosis of tumor cells. Ferroptosis and the regulation of Survivin protein can synergistically downregulate the expression of HSPs, thereby reducing the photothermal resistance of tumor cells in PTT. Additionally, FCRM caused significant tumor magnetic resonance contrast enhancement, which aided in the accurate diagnosis of tumors. Therefore, FCRM has great potential in achieving targeted magnetic resonance imaging and dual regulation of HSPs for ferroptosis-gene enhanced tumor PTT.
研究领域:
肿瘤学
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纳米医学
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基因编辑
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产品关联
DOI
PubMed
高影响因子
Bioengineered
·
2022
·
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
·
医学
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产品关联
DOI
PubMed
高影响因子
DRUG METABOLISM AND DISPOSITION
·
2020
·Vol. 48
·期 11
·1129-1136
·
IF 3.231
Jian Lu,Ang Chen,Xinrun Ma,Xuyang Shang,Yuanjin Zhang,Yuanqing Guo,Mingyao Liu,Xin Wang
Cytochrome P450 2J2 (CYP2J2) enzyme attracts more attention because it not only metabolizes clinical drugs but also mediates the biotransformation of important endogenous substances and the regulation of physiologic function. Although CYP2J2 is very important, few animal models are available to study its function in vivo. In particular, a CYP2J gene knockout (KO) rat model for drug metabolism and pharmacokinetics is not available. In this report, the CRISPR/Cas9 technology was used to delete rat CYP2J3/10, the orthologous genes of CYP2J2 in humans. The CYP2J3/10 KO rats were viable and fertile and showed no off-target effect. Compared with wild-type (WT) rats, the mRNA and protein expression of CYP2J3/10 in liver, small intestine, and heart of KO rats were completely absent. At the same time, CYP2J4 mRNA expression and protein expression were significantly decreased in these tissues. Further in vitro and in vivo metabolic studies of astemizole, a typical substrate of CYP2J, indicated that CYP2J was functionally inactive in KO rats. The heart function indexes of WT and KO rats were also measured and compared. The myocardial enzymes, including creatine kinase–muscle brain type (CK-MB), creatine kinase (CK), and CK-MB/CK ratio, of KO rats increased by nearly 140%, 80%, and 60%, respectively. In conclusion, this study successfully developed a new CYP2J3/10 KO rat model, which is a useful tool to study the function of CYP2J in drug metabolism and cardiovascular disease.
研究领域:
药理学
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分子生物学
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遗传学
✓ 已验证
DOI
PubMed
高影响因子
MICROCHIMICA ACTA
·
2024
·Vol. 191
·期 8
·1-9
·
IF 5.3
Zhou Yanmei,Che Shengjun,Wang Zhili,Zhang Xiaoru,Yuan Xunyi
An electrochemical sensor assisted by primer exchange reaction (PER) and CRISPR/Cas9 system (PER-CRISPR/Cas9-E) was established for the sensitive detection of dual microRNAs (miRNAs). Two PER hairpin (HP) were designed to produce a lot of extended PER products, which could hybridize with two kinds of hairpin probes modified on the electrode and initiate the cleavage of two CRISPR/Cas9 systems guided by single guide RNAs (sgRNAs) with different recognition sequences. The decrease of the two electrochemical redox signals indicated the presence of dual-target miRNAs. With the robustness and high specificity of PER amplification and CRISPR/Cas9 cleavage system, simultaneous detection of two targets was achieved and the detection limits for miRNA-21 and miRNA-155 were 0.43 fM and 0.12 fM, respectively. The developed biosensor has the advantages of low cost, easy operation, and in-situ detection, providing a promising platform for point-of-care detection of multiple miRNAs. Graphical abstract
研究领域:
生物化学
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分子生物学
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分析化学
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生物技术
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产品关联
DOI
PubMed
高影响因子
Synthetic and Systems Biotechnology
·
2024
·
IF 4.4
Bing Fu,Meng Chen,Xianfeng Bao,Jiajie Lu,Zhiwen Zhu,Fuyao Guan,Chuyang Yan,Peize Wang,Linglin Fu,Ping Yu
Vitamin B 2 is an essential water-soluble vitamin. For most prokaryotes, a bifunctional enzyme called FAD synthase catalyzes the successive conversion of riboflavin to FMN and FAD. In this study, the plasmid pNEW-AZ containing six key genes for the riboflavin synthesis was transformed into strain R2 with the deleted FMN riboswitch, yielding strain R5. The R5 strain could produce 540.23 ± 5.40 mg/L riboflavin, which was 10.61 % higher than the R4 strain containing plasmids pET-AE and pAC-Z harboring six key genes. To further enhance the production of riboflavin, homology matching and molecular docking were performed to identify key amino acid residues of FAD synthase. Nine point mutation sites were identified. By comparing riboflavin kinase activity, mutations of T203D and N210D, which respectively decreased by 29.90 % and 89.32 % compared to wild-type FAD synthase, were selected for CRISPR/Cas9 gene editing of the genome, generating engineered strains R203 and R210. pNEW-AZ was transformed into R203, generating R6. R6 produced 657.38 ± 47.48 mg/L riboflavin, a 21.69 % increase compared to R5. This study contributes to the high production of riboflavin in recombinant E. coli BL21.
研究领域:
生物工程
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分子生物学
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DOI
PubMed
高影响因子
MICROCHIMICA ACTA
·
2019
·Vol. 186
·期 4
·1-8
·
IF 5.479
Chang Weidan,Liu Weipeng,Liu Ying,Zhan Fangfang,Chen Huifang,Lei Hongtao,Liu Yingju
A colorimetric method is presented for the detection of specific nucleotide sequences in plant pathogens. It is based on the use of CRISPR/Cas9-triggered isothermal amplification and gold nanoparticles (AuNPs) as optical probes. The target DNA was recognized and broken up by a given Cas9/sgRNA complex. After isothermal amplification, the product was hybridized with oligonucleotide-functionalized AuNPs. This resulted in the aggregation of AuNPs and a color change from wine red to purple. The visual detection limit is 2 pM of DNA, while a linear relationship exists between the ratio of absorbance at 650 and 525 nm and the DNA concentration in the range from 0.2 pM to 20 nM. In contrast to the previous CRISPR-based amplification platforms, the method has significantly higher specificity with the single-base mismatch and can be visually read out. It was successfully applied to identify the Phytophthora infestans genomic DNA.
Schematic presentation of a colorimetric method for detection of Phytophthora infestans genomic DNA based on CRISPR/Cas9-triggered isothermal amplification. The Cas9 endonuclease cleaves DNA at the design site and the color changes from red to purple with increasing target DNA concentration.
研究领域:
分子生物学
·
植物病理学
·
纳米生物技术
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产品关联
DOI
PubMed
高影响因子
Acta Pharmaceutica Sinica B
·
2024
·
IF 14.7
Xiaoyu Wu,Zijun Bai,Hui Wang,Hanqing Wang,Dahai Hou,Yunzhu Xu,Guanqun Wo,Haibo Cheng,Dongdong Sun,Weiwei Tao
Copper-based nanomaterials demonstrate promising potential in cancer therapy. Cu + efficiently triggers a Fenton-like reaction and further consumes the high level of glutathione, initiating chemical dynamic therapy (CDT) and ferroptosis. Cuproptosis, a newly identified cell death modality that represents a great prospect in cancer therapy, is activated. However, active homeostatic systems rigorously keep copper levels within cells exceptionally low, which hinders the application of cooper nanomaterials-based therapy. Herein, a novel strategy of CRISPR-Cas9 RNP nanocarrier to deliver cuprous ions and suppress the expression of copper transporter protein ATP7A for maintaining a high level of copper in cytoplasmic fluid is developed. The Cu 2 O and organosilica shell would degrade under the high level of glutathione and weak acidic environment, further releasing RNP and Cu + . The liberated Cu + triggered a Fenton-like reaction for CDT and partially transformed to Cu 2+ , consuming intracellular GSH and initiating cuproptosis and ferroptosis efficiently. Meanwhile, the release of RNP effectively reduced the expression of copper transporter ATP7A, subsequently increasing the accumulation of cooper and enhancing the efficacy of CDT, cuproptosis, and ferroptosis. Such tumor microenvironment responsive multimodal nanoplatform opens an ingenious avenue for colorectal cancer therapy based on gene editing enhanced synergistic cuproptosis/CDT/ferroptosis.
研究领域:
生物技术
·
纳米技术
·
肿瘤学
·
基因编辑
✓ 已验证
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）