TALANTA
·
2021
·
IF 6.057
Xianfeng Wang,Xiaolong Chen,Chengxiang Chu,Yuanyi Deng,Mei Yang,Danqun Huo,Faliang Xu,Changjun Hou,Jiayi Lv
Accurate and effective detection of single-stranded nucleic acids is vital in both disease diagnosis and pathological studies. Hence, we develop a PAMmer-assisted CRISPR/Cas9 system mediated G4-EXPAR (Cas-G4EX) strategy for site-specific detection of ssRNA and ssDNA . PAMmer-assisted CRISPR/Cas9 executes the site-specific cleavage of target ssRNA or ssDNA and released product fragment with the desired sequence at the 3′-terminal. This fragment serves as a primer to activate subsequent sequence-dependent exponential amplification reaction (EXPAR). The G-rich EXPAR products assembles with hemin to form a G-Quadruplex (G4/hemin). G4/hemin catalyzes ABTS-H 2 O 2 system with the appearance of vivid green color, realizing naked-eye analysis. Cas-G4EX integrates the superiority of CRISPR/Cas9 and EXPAR, presenting outstanding site-specific recognition and high-performance amplification efficiency. Meanwhile, the programmability of CRISPR/Cas9 system makes the proposed method become a universal detection paradigm for any ssRNA or ssDNA. Cas-G4EX assay shows the linear relationship from 250 aM to 2.5 nM for ssRNA detection with the actual LOD of 250 aM, and that ranges from 100 aM to 1 nM for ssDNA detection with the actual LOD of 100 aM. Additionally, the acceptable recoveries of 101.48%–109.61% for ssRNA and 93.25%–111.98% for ssDNA in real detection of human serum are obtained for detection of single-strand nucleic acid in real samples. Cas-G4EX also exhibits the excellent discrimination for single-base mutation of single-stranded nucleic acids. Therefore, Cas-G4EX assay provides a promising platform in the applications of molecular diagnosis and pathological analysis.
研究领域:
生物技术
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分子生物学
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诊断医学
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病理学
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产品关联
DOI
PubMed
高影响因子
PEST MANAGEMENT SCIENCE
·
2024
·
IF 3.8
Yun Wang,Hui-Qian Xu,Hong-Liang Han,Dong Chen,Hongbo Jiang,Guy Smagghe,Jin-Jun Wang,Dong Wei
BACKGROUND The melon fly, Zeugodacus cucurbitae (Coquillett), is an invasive Tephritidae pest with robust fertility. The male accessory glands (MAGs) form a vital organ that ensures insect reproductive efficiency. Most of the secreted proteins by MAGs exhibit a male bias expression. Takeout, one of these proteins, is abundantly present in the MAGs of many insects. RESULTS In this study, we identified 32 takeout genes in Z. cucurbitae . The phylogenetic analysis and multiple sequence alignment results showed that Zctakeout1 is the most related homolog to the MAGs-specific takeout in Tephritidae. The real-time quantitative PCR results showed that Zctakeout1 was exclusively expressed in the male adult stage, and its expression level gradually increased with the increase in age and then remained stable at the sexually matured stage. The distribution among tissues demonstrated the specific expression of Zctakeout1 in the MAGs, and fluorescence immunohistochemical results confirmed the presence of Zctakeout1 in close proximity to binuclear cells of the mesoderm epidermal MAGs. In continuation, CRISPR/Cas9-mediated genome editing was employed, resulting in successfully generating a homozygous strain with an +8 bp insertion. The mating experiments with the Zctakeout1 −/− males resulted in significant reductions in both the mating rate and egg production of females. CONCLUSION These findings prove that the MAGs-specific Zctakeout1 is essential in regulating fecundity in female Z. cucurbitae fruit flies. Our data suggests its utilization in future essential insect-specific gene-directed sterility insect technique (SIT) by the genetic manipulation to keep these important Tephritidae populations under control. © 2024 Society of Chemical Industry.
研究领域:
昆虫学
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遗传学
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分子生物学
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生物技术
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产品关联
DOI
PubMed
高影响因子
Genes
·
2025
·Vol. 16
·期 2
·217
·
IF 2.8
Haiyan Shu,Aiping Luan,Hidayat Ullah,Junhu He,You Wang,Chengjie Chen,Qing Wei,Rulin Zhan,Shenghe Chang
Background/Objectives: CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats)-associated protein 9 is now widely used in agriculture and medicine. Off-target effects can lead to unexpected results that may be harmful, and these effects are a common concern in both research and therapeutic applications. Methods: In this study, using pineapple as the gene-editing material, eighteen target sequences with varying numbers of PAM (Protospacer-Adjacent Motif) sites were used to construct gRNA vectors. Fifty mutant lines were generated for each target sequence, and the off-target rates were counted. Results: Selecting sequences with multiple flanking PAM sites as editing targets resulted in a lower off-target rate compared to those with a single PAM site. Target sequences with two 5′-NGG (“N” represents any nucleobase, followed by two guanine “G”) PAM sites at the 3′ end exhibited greater specificity and a higher probability of binding with the Cas9 protein than those only with one 5′-NGG PAM site at the 3′ end. Conversely, although the target sequence with a 5′-NAG PAM site (where “N” is any nucleobase, followed by adenine “A” and guanine “G”) adjacent and upstream of an NGG PAM site had a lower off-target rate compared to sequences with only an NGG PAM site, their off-target rates were still higher than those of sequences with two adjacent 5′-NAG PAM sites. Among the target sequences of pineapple mutant lines (AcACS1,AcOT5,AcCSPE6,AcPKG11A), more deletions than insertions were found. Conclusions: We found that target sequences with multiple flanking PAM sites are more likely to bind with the Cas9 protein and induce mutations. Selecting sequences with multiple flanking PAM sites as editing targets can reduce the off-target effects of the Cas9 enzyme in pineapple. These findings provide a foundation for improving off-target prediction and engineering CRISPR-Cas9 complexes for gene editing.
CAS / MDL:
研究领域:
植物学
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遗传学
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分子生物学
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产品关联
DOI
PubMed
高影响因子
Forests
·
2024
·Vol. 15
·期 7
·1157
·
IF 2.4
Huihui Liu,Lin Chen,Qinghua Ma,Xinghong Wang,Sufang Zhang,Xuguo Zhou
Hox genes, specifically the bithorax complex (ubx,abdominal-a, andabdominal-b), play a crucial role in specifying posterior abdominal development and serve as key regulators of germline gene development in insects. However, the function of the bithorax complex in the pine caterpillar moth,Dendrolimus punctatus, a major pine tree defoliator in China, remains largely unknown. Specifically,Abdominal-A(Abd-a) controls regional variation in abdominal segmentation in model insects such asDrosophilaandTribolium; however, its role inD. Punctatusremains unexplored. In this study, CRISPR/Cas9 was used to functionally characterizeAbd-ainD. punctatus. Two target sites were selected, and the genotypes and phenotypes of the G0 and G1 generations were evaluated. Our findings indicate that knocking outAbd-aled to an abnormality in the posterior segments A2–A7, as well as the loss of appendages, mainly prolegs, and affected the thoracic T3 segmentation as well as wing development. Moreover, mutation inAbd-aalso impacted anal and reproductive development. Taken together, these results demonstrate thatDpAbd-ais essential for embryonic and reproductive development inD. punctatusand could be a promising target for genetic control of this devastating conifer defoliator.
研究领域:
昆虫学
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遗传学
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分子生物学
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产品关联
DOI
高影响因子
Aquaculture and Fisheries
·
2026
·
IF 5
Weizhong Li,Wenyao Lv,Tianyang Sun,Qian Cong,Weipeng Lian,Tao Sheng,Hongquan Zhang,Qiaozhen Yu,Yuxuan Chen,Xinzhan Meng,Jianfeng Ren,Lang Gui,Junqiang Qiu,Guiling Wang,Xiaoyan Xu,Jiale Li
The transforming growth factor-beta (TGF-beta) signaling pathway is a central regulator of skeletal muscle growth in vertebrates, but the function of Acvr1b (activin receptor type 1B) remains poorly characterized in teleosts. Here, we used CRISPR/Cas9 to disrupt Acvr1b in grass carp ( Ctenopharyngodon idella ) and evaluated growth performance, muscle histology, and myog enic gene expression. Cas9 protein and sgRNAs targeting exons 2 and 3 were co-injected into fertilized eggs, and edited juveniles were identified by T7E1 assay and Sanger sequencing. Acvr1b -edited fish showed significantly improved growth relative to wild-type controls, including a 25.8% increase in body weight and a 7.39% increase in body length. qRT-PCR revealed reduced Acvr1b expression in all examined tissues. Consistent with enhanced muscle growth, mstn was downregulated, myog was upregulated, and myod was unchanged in edited fish. Histological analysis showed increased muscle fiber size and density, indicating that both hypertrophy and hyperplasia contributed to the growth advantage. Together, these findings identify Acvr1b as a promising target for genetic improvement in grass carp aquaculture.
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产品关联
DOI
高影响因子
Frontiers in Bioengineering and Biotechnology
·
2022
·
IF 6.064
Wei Chao-Gang,Zhang Rui,Wei Lan-Yi,Pan Peng,Zu He,Liu Ya-Zhen,Wang Yong,Shen Jun-Kang
Introduction: Erythropoietin producing hepatocyte receptor A2 (EphA2) is widely presented in the tumor cells, closely related to tumor cell migration, not cell apoptosis and proliferation. Based on its high expression in castration-resistant prostate cancer (CRPC), we herein develop a CRISPR-Cas9-based genome-editing nanomedicine to target erythropoietin producing hepatocyte receptor A2 for the treatment of castration-resistant prostate cancer.Methods: To this end, TAT was designed to stabilize the distribution of calcium, and then bound to ribonucleoprotein (RNP) to form nanoparticles RNP@CaP-TAT.Results: This nanoparticle has a simple synthesis process with good biocompatible, to achieve the knockout of tumor cells (PC-3) targeting erythropoietin producing hepatocyte receptor A2 gene and to effectively suppress the migration of tumor cells.Discussion: This delivery genome editing system provides a promising gene therapy strategy for the treatment of castration-resistant prostate cancer, showing good potential against castration-resistant prostate cancer tumor metastasis. In addition, it can be extended to other types of cancer with highly heterogeneous gene expression.
研究领域:
纳米技术
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癌症生物学
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基因编辑
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分子生物学
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生物医学工程
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DOI
PubMed
高影响因子
Journal of Fungi
·
2023
·Vol. 9
·期 8
·863
·
IF 4.7
Yinggu Wu,Xinyu Li,Li Dong,Tong Liu,Zhengbin Tang,Runmao Lin,Justice Norvienyeku,Mengyu Xing
The litchi downy blight disease of litchi caused byPeronophythora litchiiaccounts for severe losses in the field and during storage. While ample quantitative studies have shown that 6-pentyl-2H-pyran-2-one (6PP) possesses antifungal activities against multiple plant pathogenic fungi, the regulatory mechanisms of 6PP-mediated inhibition of fungal pathogenesis and growth are still unknown. Here, we investigated the potential molecular targets of 6PP in the phytopathogenic oomycetesP. litchiithrough integrated deployment of RNA-sequencing, functional genetics, and biochemical techniques to investigate the regulatory effects of 6PP againstP. litchii. Previously we demonstrated that 6PP exerted significant oomyticidal activities. Also, comparative transcriptomic evaluation ofP. litchiistrains treated with 6PP Revealed significant up-regulations in the expression profile of TOR pathway-related genes, includingPlCytochrome Cand the transcription factorsPlYY1. We also noticed that 6PP treatment down-regulated putative negative regulatory genes of the TOR pathway, includingPlSpm1andPlrhoH12inP. litchii. Protein-ligand binding analyses revealed stable affinities between PlYY1, PlCytochrome C, PlSpm1, PlrhoH12 proteins, and the 6PP ligand. Phenotypic characterization ofPlYY1targeted gene deletion strains generated in this study using CRISPR/Cas9 and homologous recombination strategies significantly reduced the vegetative growth, sporangium, encystment, zoospore release, and pathogenicity ofP. litchii. These findings suggest that 6PP-mediated activation ofPlYY1expression positively regulates TOR-related responses and significantly influences vegetative growth and the virulence ofP. litchii. The current investigations revealed novel targets for 6PP and underscored the potential of deploying 6PP in developing management strategies for controlling the litchi downy blight pathogen.Keywords:P. litchii;6-pentyl-2H-pyran-2-one (6PP);TOR pathway;CRISPR/Cas 9;PlYY1
研究领域:
植物病理学
·
分子生物学
·
真菌学
✓ 已验证
DOI
PubMed
高影响因子
Antibiotics-Basel
·
2026
·Vol. 15
·期 2
·202
·
IF 5.5
Zhiping Hou,Mengliu Peng,Xiaozhi Ju,Yaqi Sun,Liping Du,Ye Liang,Shu-Shan Gao
Background: The fungusAcremonium chrysogenumis crucial for producing cephalosporin antibiotics. While CRISPR/Cas9 has been developed for this species, it has not been applied to first-line industrial strains, to the best of our knowledge. For example, engineering industrialA. chrysogenumfor overproducing deacetoxycephalosporin C (DAOC, an important precursor for clinically used cephalosporin antibiotics) is currently often a multi-step and inefficient process. Methods: Here, we applied CRISPR/Cas9 to create a DAOC overproducer in a single step. Our method uses a donor template to simultaneously delete and overexpress genes, offering a simple, efficient, and time-saving solution. Results: Furthermore, through methodological optimization, the final homozygous multigene-edited strain achieved an industrial-scale DAOC titer of 12.4 ± 0.2 g/L, representing a significant improvement over the initial edited strain (7.2 ± 0.22 g/L). Conclusions: This demonstrates that CRISPR/Cas9 can effectively edit industrialA. chrysogenum, providing a strategy to enhance the production of other cephalosporin precursors.
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产品关联
DOI
PubMed
高影响因子
Science Advances
·
2019
·
IF 12.804
Yongchun Pan,Jingjing Yang,Xiaowei Luan,Xinli Liu,Xueqing Li,Jian Yang,Ting Huang,Lu Sun,Yuzhen Wang,Youhui Lin,Yujun Song
As an RNA-guided nuclease, CRISPR-Cas9 offers facile and promising solutions to mediate genome modification with respect to versatility and high precision. However, spatiotemporal manipulation of CRISPR-Cas9 delivery remains a daunting challenge for robust effectuation of gene editing both in vitro and in vivo. Here, we designed a near-infrared (NIR) light–responsive nanocarrier of CRISPR-Cas9 for cancer therapeutics based on upconversion nanoparticles (UCNPs). The UCNPs served as “nanotransducers” that can convert NIR light (980 nm) into local ultraviolet light for the cleavage of photosensitive molecules, thereby resulting in on-demand release of CRISPR-Cas9. In addition, by preparing a single guide RNA targeting a tumor gene (polo-like kinase-1), our strategies have successfully inhibited the proliferation of tumor cell via NIR light–activated gene editing both in vitro and in vivo. Overall, this exogenously controlled method presents enormous potential for targeted gene editing in deep tissues and treatment of a myriad of diseases.
研究领域:
遗传学
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纳米技术
·
癌症研究
·
分子生物学
✓ 已验证
DOI
PubMed
高影响因子
BIOMEDICINE & PHARMACOTHERAPY
·
2022
·
IF 7.419
Qingxia Zuo,Wanqing Xu,Yanbin Wan,Dongyan Feng,Changsheng He,Cailing Lin,Dongchao Huang,Feng Chen,Liya Han,Qi Sun,Dong Chen,Hongli Du,Lizhen Huang
CRISPR/Cas9 has allowed development of better and easier-to-use ADME models than traditional methods by complete knockout or knock-in of genes. However, gene editing in HepaRG cells remains challenging because long-term monoclonal cultivation may alter their differentiation capacity to a large extent. Here, CRISPR/Cas9 was used to generate a CYP3A4-T2A-luciferase knock-in HepaRG subclone by Cas9-mediated homologous recombination and monoclonal cultivation. The knock-in HepaRG-#9 subclone retained a similar differentiation potential to wildtype HepaRG cells (HepaRG-WT). To further improve differentiation and expand the applications of knock-in HepaRG cells, two optimized differentiation procedures were evaluated by comparison with the standard differentiation procedure using the knock-in HepaRG-#9 subclone and HepaRG-WT. The results indicated that addition of forskolin (an adenylate cyclase activator) and SB431542 (a TGF-β pathway inhibitor) to the first optimized differentiation procedure led to better differentiation consequence in terms of not only the initiation time for differentiation and morphological characterization, but also the mRNA levels of hepatocyte-specific genes. These data may contribute to more extensive applications of genetically modified HepaRG cells in ADME studies.
研究领域:
药理学
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分子生物学
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基因工程
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产品关联
DOI
PubMed
高影响因子
Nature Nanotechnology
·
2025
·
IF 34.9
Zhao Kai,Yan Yu,Jin Xiao-Kang,Pan Ting,Zhang Shi-Man,Yang Chi-Hui,Rao Zhi-Yong,Zhang Xian-Zheng
Chemoresistance and immunosuppression are common obstacles to the efficacy of chemo-immunotherapy in colorectal cancer (CRC) and are regulated by mitochondrial chaperone proteins. Here we show that the disruption of the tumour necrosis factor receptor-associated protein 1 ( TRAP1 ) gene, which encodes a mitochondrial chaperone in tumour cells, causes the translocation of cyclophilin D in tumour cells. This process results in the continuous opening of the mitochondrial permeability transition pore, which enhances chemotherapy-induced cell necrosis and promotes immune responses. On the basis of this discovery we developed an oral CRISPR–Cas9 delivery system based on zwitterionic and polysaccharide polymer-coated nanocomplexes that disrupts the TRAP1 gene in CRC. This system penetrates the intestinal mucus layer and undergoes epithelial transcytosis, accumulating in CRC tissues. It enhances chemotherapeutic efficacy by overcoming chemoresistance and activating the tumour immune microenvironment in orthotopic, chemoresistant and spontaneous CRC models, with remarkable synergistic antitumour effects. This oral CRISPR–Cas9 delivery system represents a promising therapeutic strategy for the clinical management of CRC.
研究领域:
肿瘤学
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分子生物学
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基因编辑
·
纳米医学
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产品关联
DOI
PubMed
高影响因子
PARASITOLOGY RESEARCH
·
2019
·Vol. 118
·期 3
·935-944
·
IF 2.067
Ye Shu,Xia Ningbo,Zhao Pengfei,Yang Jichao,Zhou Yanqin,Shen Bang,Zhao Junlong
Toxoplasma gondii is a ubiquitous parasitic protozoan infecting humans and a wide variety of animals. Fast-replicating tachyzoites during acute infection and slowly growing bradyzoites during chronic infection are the two basic forms of T. gondii in intermediate hosts. Interconversion between the two contributes to the transmission and pathogenesis of this parasite. Secretory micronemal proteins are thought to mediate interactions with host cells and facilitate parasite invasion, therefore the majority of them are highly expressed in tachyzoites. Micronemal protein 13 (MIC13) is unique in that its expression is low in tachyzoites and is upregulated under bradyzoite-inducing conditions. Previous attempts to disrupt this gene were not successful, implying that it may play critical roles during parasite growth. However, in this study, MIC13 was successfully disrupted in type 1 strain RH and type 2 strain ME49 using CRISPR/Cas9-mediated gene disruption techniques. Consistent with its low expression in tachyzoites and increased expression under stress or bradyzoite-inducing conditions, MIC13-inactivated mutants displayed normal growth, host cell invasion, intracellular replication, and egress, as well as acute virulence at the tachyzoite stage. However, under stress conditions, such as high pH or oxygen limitation, MIC13-disrupted parasites showed significantly slower growth rates compared to the parental strains, suggesting that it is required for optimal parasite growth under bradyzoite-inducing or stress conditions. This is the first micronemal protein reported to have such expression pattern and function modes, which expands our understanding of the diverse functions of micronemal proteins.
研究领域:
寄生虫学
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分子生物学
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传染病学
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DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）