BIOTECHNOLOGY AND BIOENGINEERING
·
2021
·Vol. 118
·期 5
·2053-2066
·
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
·
医学诊断
·
基因编辑
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ANALYST
·
2020
·Vol. 145
·期 10
·3569-3574
·
IF 3.978
Rui Guo,Shanwen Hu,Zonghua Wang
CRISPR/Cas9 has become a powerful tool for genomic manipulation, and the evaluation of the Cas9 activity is essential for the precise control of the CRISPR system. Herein, we develop a point-of-care platform for the rapid and visible determination of the Cas9 activity. A bipolar electrochemiluminescence (ECL) biosensor platform comprises an Au electric circuit fabricated on a glass substrate and the PDMS layer acts as a reservoir. DNA probes are then modified on electrodes and hybridization chain reaction (HCR) is applied to introduce ferrocene-labeled DNA. The CRISPR/Cas9 system can recognize the immobilized DNA probes and cleave them from the electrode surface as well as the labeled H1 and H2. Therefore, the cleavage activity is closely related to the labeled ferrocene. The modified electrode is then embodied in a closed bipolar system such that the oxidation reaction on sensing poles could be detected by the anodic ECL reactions on isolated luminescent poles due to charge balance, leading to the changes in ECL signals. This portable, integrated and convenient platform may become a meaningful tool for the discovery of a new CRISPR/Cas9 system or additional Cas9 inhibitors.
研究领域:
生物技术
·
分子生物学
·
分析化学
✓ 已验证
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
·
发酵工程
·
食品科学
✓ 已验证
产品关联
DOI
高影响因子
Nanoscale
·
2017
·Vol. 10
·期 3
·1063-1071
·
IF 7.367
Huahua Yue,Xiaoming Zhou,Meng Cheng,Da Xing
Direct cellular delivery of CRISPR/Cas9 complexes is of great significance for genome editing and other recently developed applications, such as gene expression regulation and RNA/DNA imaging. Here, we first constructed a graphene oxide (GO)-polyethylene glycol (PEG)-polyethylenimine (PEI) nanocarrier for the delivery of high-molecular-weight Cas9/single-guide RNA (sgRNA) complexes for endocytosis, endosomal escape, nuclear entry, and gene editing. The results demonstrate that the nanocarrier can be used successfully for efficient gene editing in human AGS cells with an efficiency of ∼39%. The results also show that this nanocarrier can protect sgRNA from enzymatic degradation, thus exhibiting extremely high stability, which is critical for future in vivo applications. Thus, this GO-mediated Cas9/sgRNA delivery system has potential as a new approach for biomedical research and targeted gene engineering applications.
研究领域:
生物技术
·
遗传学
·
纳米医学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
CHEMICAL ENGINEERING JOURNAL
·
2024
·
IF 13.3
Liansheng Fan,Suchen Qu,Jing Qian,Gaoyu Shi,Qiqing Huang,Yanni Song,Yu Ji,Quli Fan,Xin Han
Immune checkpoint blockade (ICB) has shown significant progress in treating triple negative breast cancer (TNBC). However, the standalone effectiveness of ICB is modest, offering patients only slight benefits. In this report, we utilize a hypoxia-responsive duplex genome editing strategy to target tumor cell programmed cell death ligand 1 (PD-L1) and protein tyrosine phosphatase non-receptor type 2 (PTPN2), which is synergized with photothermal therapy (PTT) to amplify the therapeutic effect of TNBC. In our approach, the melanin nanoparticles (MNP) serve as the delivery platform for transporting the CRISPR-Cas PTPN2/PD-L1 (Cas-TD) system, which is cross-linked by a hypoxic-sensitive Azo linker and then further modified with tumor homing phenylboronic acid (PBA), resulting in a stimuli-responsive multifunctionality nanotheranostic platform (MPA@Cas-TD@PP). Aided by the guidance of PBA, nanoparticles selectively accumulate at tumor and then disintegrate with enhanced intracellular penetration. Importantly, PTT initiates the immunogenic cell death (ICD), turning the “cold” tumor into a “hot” one, laying the foundation for subsequent ICB. Inhibiting PTPN2 and PD-L1 can synergistically sensitize tumors to immunotherapy, which exhibit magnified immunotherapy effects by increasing activated cytotoxic CD8 + T cells and enhancing interferon-γ (IFN-γ)-mediated effects. In summary, this multifunctional nanotherapeutic platform provides a promising treatment approach for synergistic phototherapy and immunotherapy in TNBC.
研究领域:
肿瘤学
·
基因编辑
·
纳米医学
·
免疫治疗
✓ 已验证
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
·
诊断学
·
生物医学工程
✓ 已验证
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
·
分子生物学
·
风湿病学
✓ 已验证
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
·
纳米技术
·
细胞生物学
·
基因组学
✓ 已验证
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
·
纳米医学
·
基因编辑
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Frontiers in Cellular and Infection Microbiology
·
2019
·
IF 3.518
Liu Qiushi,Lei Jing,Kadowaki Tatsuhiko
Two trypanosomatid species, Lotmaria passim and Crithidia mellificae, have been shown to parasitize honey bees to date. L. passim appears to be more prevalent than C. mellificae and specifically infects the honey bee hindgut. Although the genomic DNA has been sequenced, the effects of infection on honey bee health and colony are poorly understood. To identify the genes that are important for infecting honey bees and to understand their functions, we applied the CRISPR/Cas9 system to establish a method to manipulate L. passim genes. By electroporation of plasmid DNA and subsequent selection by drug, we first established an L. passim clone expressing tdTomato or Cas9. We also successfully disrupted the endogenous miltefosine transporter and tyrosine aminotransferase genes by replacement with drug (hygromycin) resistant gene using the CRISPR/Cas9-induced homology-directed repair pathway. The L. passim clone expressing fluorescent marker, as well as the simple method for editing specific genes, could become useful approaches to understand the underlying mechanisms of honey bee-trypanosomatid parasite interactions.
研究领域:
昆虫学
·
分子生物学
·
寄生虫学
·
遗传学
✓ 已验证
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
·
遗传学
·
医学
✓ 已验证
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
·
分子生物学
·
遗传学
✓ 已验证
DOI
PubMed
高影响因子
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