VETERINARY MICROBIOLOGY
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2024
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IF 2.4
Le-le Liu,You-qin Yin,Kai-xiong Ma,Jin-chao Xing,Xing-xing Ren,Jin-yu Huang,Ming Liao,Wen-bao Qi,Li-hong Huang
Japanese encephalitis virus (JEV) is a pathogen with a substantial impact on both livestock and human health. However, the critical host factors in the virus life cycle remain poorly understood. Using a library comprising 123411 small guide RNAs (sgRNAs) targeting 19050 human genes, we conducted a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based screen to identify essential genes for JEV replication. By employing knockout or knockdown techniques on genes, we identified eleven human genes crucial for JEV replication, such as prolactin releasing hormone receptor (PRLHR), activating signal cointegrator 1 complex subunit 3 (ASCC3), acyl-CoA synthetase long chain family member 3 (ACSL3), and others. Notably, we found that PRLHR knockdown blocked the autophagic flux, thereby inhibiting JEV infection. Taken together, these findings provide effective data for studying important host factors of JEV replication and scientific data for selecting antiviral drug targets.
研究领域:
病毒学
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分子生物学
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基因组学
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DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
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2022
·Vol. 23
·期 24
·16082
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IF 6.208
Zhaoyang Li,Muhammad Junaid Rao,Jiaying Li,Yanting Wang,Peng Chen,Hua Yu,Chongjian Ma,Lingqiang Wang
Pectin is one of the constituents of the cell wall, distributed in the primary cell wall and middle lamella, affecting the rheological properties and the cell wall stickiness. Pectin methylesterase (PME) and pectin methylesterase inhibitor (PMEI) are the most important factors for modifying methyl esterification. In this study, 45PMEIgenes from rice (Oryza sativaL.) were screened by bioinformatics tools, and their structure, motifs, cis-acting elements in the promoter region, chromosomal distribution, gene duplication, and phylogenetic relationship were analyzed. Furthermore, CRISPR/Cas9 was used to edit theOsPMEI12(LOC_Os03G01020) and two mutantpmei12lines were obtained to explore the functions ofOsPMEIin plant growth and development, and under cadmium (Cd) stress. Compared to wild type (WT) Nipponbare, the second inverted internodes of the mutant plants shortened significantly, resulting in the reduction in plant height at mature stage. The seed setting rate, and fresh and dry weights of the mutants were also decreased in mutant plants. In addition, the pectin methylation ofpmei12lines is decreased as expected, and the pectin content of the cell wall increased at both seedling and maturity stages; however, the cellulose and hemicellulose increased only at seedling stage. Interestingly, the growth of thepmei12lines was better than the WT in both normal conditions and under two phytohormone (GA3and NAA) treatments at seedling stage. Under Cd stress, the fresh and dry weights were increased inpmei12lines. These results indicated thatOsPMEI12was involved in the regulation of methyl esterification during growth, affected cell wall composition and agronomic traits, and might play an important role in responses to phytohormones and stress.Keywords:Oryza sativaL.;pectin methylesterase inhibitor;cell wall;plant growth regulators;Cd stress
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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DOI
PubMed
高影响因子
Frontiers in Bioengineering and Biotechnology
·
2025
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IF 4.8
Yunpeng Wu,Jianhua Zheng,Jingqing Chen,Tianqi Sun,Tianqi Luan,Yan Li,Yunzhi Fa,Yefeng Qiu,Rui Zhang
Introduction Human butyrylcholinesterase (hBChE) is a promising bioscavenger against organophosphorus (OP) nerve agents and pesticides. However, low homology-directed repair (HDR) efficiency in CRISPR/Cas9-mediated genome editing limits precise transgene integration in large animals. Methods To improve HDR, we optimized donor structure for targeted integration of hBChE into the goat FGF5 locus using CRISPR/Cas9. Correctly edited goat fibroblast clones were identified by PCR and sequencing. A homozygous clone with reverse-oriented integration was used as a donor for somatic cell nuclear transfer (SCNT), followed by embryo transfer. Offspring were analyzed for genomic integration and transgene expression. Results Reverse-oriented donors significantly enhanced HDR efficiency compared to forward designs, with validation at the pig RAG1 locus. Edited cells stably expressed recombinant hBChE (rhBChE) and showed increased resistance to OP pesticides. SCNT produced a cloned goat expressing high rhBChE levels in the skin. Discussion Optimizing donor structure improves precise genome editing efficiency and enables robust generation of transgenic goats. This strategy advances CRISPR/Cas9-based bioreactor development for scalable production of therapeutic proteins.
研究领域:
遗传学
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分子生物学
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转基因生物技术
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生殖生物学
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DOI
PubMed
高影响因子
IMMUNITY
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2021
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IF 31.745
Run Fang,Qifei Jiang,Yukun Guan,Pengfei Gao,Rui Zhang,Zhen Zhao,Zhengfan Jiang
Summary Activation of the cyclic guanosine monophosphate (GMP)-AMP (cGAMP) sensor STING requires its translocation from the endoplasmic reticulum to the Golgi apparatus and subsequent polymerization. Using a genome-wide CRISPR-Cas9 screen to define factors critical for STING activation in cells, we identified proteins critical for biosynthesis of sulfated glycosaminoglycans (sGAGs) in the Golgi apparatus. Binding of sGAGs promoted STING polymerization through luminal, positively charged, polar residues. These residues are evolutionarily conserved, and selective mutation of specific residues inhibited STING activation. Purified or chemically synthesized sGAGs induced STING polymerization and activation of the kinase TBK1. The chain length and O -linked sulfation of sGAGs directly affected the level of STING polymerization and, therefore, its activation. Reducing the expression of Slc35b2 to inhibit GAG sulfation in mice impaired responses to vaccinia virus infection. Thus, sGAGs in the Golgi apparatus are necessary and sufficient to drive STING polymerization, providing a mechanistic understanding of the requirement for endoplasmic reticulum (ER)-to-Golgi apparatus translocation for STING activation.
研究领域:
免疫学
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细胞生物学
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分子生物学
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DOI
PubMed
高影响因子
PLANT BIOTECHNOLOGY JOURNAL
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2019
·Vol. 18
·期 7
·1550-1561
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IF 6.84
Qi Gao,Huifeng Luo,Yongping Li,Zhongchi Liu,Chunying Kang
Summary Fruit colour affects consumer preference and is an important trait for breeding in strawberry. Previously, we isolated the Reduced Anthocyanins in Petioles ( RAP ) gene encoding a glutathione S-transferase (GST) that binds anthocyanins to facilitate their transport from cytosol to vacuole in the diploid strawberry Fragaria vesca . The parent of rap was the F. vesca variety ‘Yellow Wonder’ that develops white fruit due to a natural mutation in the FveMYB10 gene. Here, we investigated the application potential of RAP in modulating fruit colours by overexpression of RAP in F. vesca and knockout of RAP in the cultivated strawberry Fragaria × ananassa . Unexpectedly, the RAP overexpression in Yellow Wonder background caused formation of red fruit. In addition, the red coloration occurs precociously at floral stage 10 and continues in the receptacle during early fruit development. Transcriptome analysis revealed that the anthocyanin biosynthesis genes were not up-regulated in RAP-ox; rap myb10 flowers at anthesis and largely inhibited at the turning stage in fruit, suggesting a coloration mechanism independent of FveMYB10 . Moreover, we used CRISPR/Cas9 to knockout RAP in cultivated strawberry which is octoploid. Six copies of RAP were simultaneously knocked out in the T0 generation leading to the green stem and white-fruited phenotype. Several T1 progeny have segregated away the CRISPR/Cas9 transgene but maintain the green stem trait. Our results indicate that enhancing the anthocyanin transport could redirect the metabolic flux from proanthocyanidin to anthocyanin production at early developmental stages of fruit and that RAP is one promising candidate gene in fruit colour breeding of strawberry.
研究领域:
植物生物学
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遗传学
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分子生物学
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农学
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DOI
PubMed
高影响因子
Advanced Science
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2026
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IF 14.1
Xuhui Fan,Ruru Zhang,Linjun Yang,Shixiong Chen,Meijuan He,Yongqiang Wang,Linjie Huang,Jianfeng Zeng,Shuwang Wu,Mingyuan Gao,Han Wang
Radiotherapy is a fundamental treatment for prostate cancer; however, its therapeutic efficacy is frequently limited by radioresistance mediated through DNA repair mechanisms and other biological factors. Although gene therapy holds promise for overcoming such resistance, identifying effective radiosensitization targets and developing efficient gene delivery systems remain practically challenging. In this study, transcriptomic analysis of radiotherapy-treated prostate cancer cells revealed a marked upregulation of DNA polymerase delta subunit 4 (POLD4), a target that has not been systematically studied. To evaluate the potential of POLD4 for overcoming radioresistance, CRISPR-Cas9-based plasmids along with ultrasmall superparamagnetic iron oxide nanoparticles (USPIONs) were encapsulated within cationic liposomes for achieving an MRI-trackable gene delivery platform (plasmid and iron oxide co-loaded liposomes, termed PIO@Lipo). Comprehensive in vitro and in vivo studies demonstrated that PIO@Lipo enabled efficient POLD4 knockdown. Furthermore, PIO@Lipo synergized with radiotherapy to induce extensive DNA damage, promote tumor cell apoptosis, and remodel the immunosuppressive microenvironment. Notably, PIO@Lipo displayed superior MRI contrast enhancement capability and passive tumor targeting ability. In conclusion, this study has identified POLD4 as a potent target for radiosensitization, capable of disrupting DNA damage-repair homeostasis through MRI-monitored gene editing, thereby offering a promising strategy to enhance the efficacy of radiotherapy in prostate cancer.
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PubMed
高影响因子
ANALYTICAL CHEMISTRY
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2020
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IF 6.785
Xuan Sun,Yu Wang,Lu Zhang,Sha Liu,Man Zhang,Jiang Wang,Baoan Ning,Yuan Peng,Jing He,Yonggang Hu,Zhixian Gao
Escherichia coli O157:H7 has been reported as an important pathogenic bacteria causing serious infection and economic loss. However, detection of Escherichia coli O157:H7 needs improvement, given its current complexity and sensitivity. Herein, we attempt to build a fluorescence sensing method to detect Escherichia coli O157:H7 with easy operation and high efficiency. The target virulence gene sequences are recognized and cleaved by the CRISPR-Cas9 system, and trigger strand displacement amplification and rolling circle amplification. After amplification reactions, massive products can hybridize with the probes, the fluorescence of which are quenched based on a metal–organic framework platform, leading to the fluorescence recovery at typical excitation/emission wavelengths of 480/518 nm. This method exhibits high sensitivity with the detection limit at 4.0 × 101 CFU mL–1 and a wide range from 1.3 × 102 CFU mL–1 to 6.5 × 104 CFU mL–1. Meanwhile, this assay also shows significant specificity and applies to practical samples with high accuracy. Therefore, our method would have great potential application in bacterial detection, food safety monitoring, or clinical diagnostics.
研究领域:
微生物学
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生物技术
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食品科学
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分子生物学
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分析化学
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DOI
PubMed
高影响因子
Advanced Healthcare Materials
·
2022
·Vol. 11
·期 16
·2201038
·
IF 11.092
Lin Huang,Mengyang Zhou,Ghulam Abbas,Chao Li,Mengmeng Cui,Xian-En Zhang,Dian-Bing Wang
Bimodal synergistic therapy produces superadditive effect for enhanced therapeutic efficacy. However, how to efficiently and simultaneously deliver several kinds of therapeutic agents is still challenging. A cancer cell membrane-derived nanocarrier (mCas9-sGNRs) is proposed for synergistic photothermal/gene therapy (PTT/GT) by efficient delivery of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) and gold nanorods (GNRs). In this approach, Cas9 proteins can be efficiently loaded inside the cell membranes (mCas9) by electrostatic interactions. Similarly, single-guide RNAs, which target survivin, can be loaded onto GNRs (sGNRs) through electrostatic interactions and encapsulated by mCas9. As a result, the nanodelivery systems present advantages in biocompatibility, homologous targeting capacity and loading efficiency of cargoes. In addition, significant antitumor effects is achieved by gene editing of survivin which induces anticancer activity and reduces heat tolerance of cancer cells caused by GNRs mediated PTT due to the downregulation of HSP70. These results indicate the nanotherapeutic platform leads to enhanced PTT/GT efficacy. Therefore, this work not only provides a general strategy to construct a versatile nanoplatform for loading and target delivery of several therapeutic cargos but will also be valuable for PTT/GT and other bimodal synergistic therapy.
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产品关联
DOI
PubMed
高影响因子
Nano Today
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2024
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IF 13.2
Jiaying Jin,Yijia Li,Feiyang Deng,Long Yang,Yuxing Lin,Canyu Yang,Wenbing Dai,Hua Zhang,Bing He,Qiang Zhang,Xueqing Wang
KRAS gene is mutated in 40 % of colorectal cancers (CRC), which induces malignant proliferation by regulating cellular nutrient metabolism and biosynthesis. It has been found that malignant proliferation of KRAS-mutant colorectal cancer relies on the upregulation of SLC25A22 protein expression, suggesting that inhibition the expression of both KRAS and SLC25A22 is a potential CRC therapeutics. Stably knocking down the oncogenic KRAS-G12V gene can achieve long-term gene therapy effects, while transient downregulation of SLC25A22, a normal functional gene most of the time, is preferred to kill tumor cells and minimize the side impact on normal cells. Here, two lipid nanoparticles (LNP) were designed to encapsulate KRAS-G12V CRISPR/Cas9 gene editing plasmids (pKRAS-LNPs) and SLC25A22 siRNA (siSLC-LNPs), respectively. Therapeutic effects of both nanoparticles alone and in combination on KRAS-G12V mutant colorectal cancer cells in vitro were first examined. The result showed that delivery of pKRAS-LNPs or siSLC-LNPs alone could effectively achieve KRAS-G12V gene editing or SLC25A22 gene silencing and inhibit tumor cell proliferation, while co-delivery of both LNPs could achieve stronger inhibition of tumor cell proliferation by inducing stronger apoptosis. Furthermore, we found that co-delivery of pKRAS-LNPs and siSLC-LNPs induced stronger apoptosis and cell proliferation inhibition compared to pKRAS&siSLC-LNPs that were constructed by pre-mixing pKRAS and siSLC and then encapsulating them. Finally, we validated that co-delivery of pKRAS-LNPs and siSLC-LNPs can achieve KRAS-G12V colorectal cancer treatment in vivo with a tumor inhibition rate of 61.15 %. In summary, the delivery vectors constructed for nucleic acids targeting KRAS and SLC25A22 achieved therapeutic targeting of KRAS-G12V colorectal cancer in vitro and in vivo .
研究领域:
肿瘤学
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分子生物学
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基因治疗
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癌症研究
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DOI
高影响因子
ADVANCED FUNCTIONAL MATERIALS
·
2023
·Vol. 33
·期 7
·2210860
·
IF 19
Chenya Zhuo,Huiming Kong,Ke Yi,Chun-Wei Chi,Jiabing Zhang,Ran Chen,Haixia Wang,Caixia Wu,Yeh-Hsing Lao,Yu Tao,Mingqiang Li
Chronic hepatitis B infection remains incurable due to the stable presence of various forms of hepatitis B virus (HBV) genome, especially the HBV covalently closed circular DNA (cccDNA). The emergence of clustered regularly interspaced short palindromic repeat (CRISPR) technology provides a new opportunity to potentially cure the HBV infection. However, the efficiency and specificity remain unsatisfactory, especially for nonviral CRISPR/Cas9 delivery. To tackle these, a liver-specific CRISPR/Cas9 magnetic nanosystem FMNP pAG333/sgXPP is constructed based on fluorinated polyethylenimine-coated magnetic nanoparticles and liver-specific ApoE.HCR.hAAT promoter-driven Cas9-T2A-EGFP plasmid with dual sgRNAs. The elaborate system enables magnetic field-induced spatially specific distribution and hepatocyte-specific promoter-driven liver-specific gene editing. Moreover, this CRISPR/Cas9 magnetic nanosystem is designed to disrupt the two conserved sites in X opening reading frame and Pol opening reading frame of the HBV genome, thereby significantly inactivating the HBV genome without showing off-target effects. Treatment with FMNP pAG333/sgXPP for 7 days reduces serum HBsAg levels by 76% with a total editing efficiency of ≈20% in the two conserved sites. Collectively, this study demonstrates spatiotemporal liver genome editing as well as the feasibility of applying a nonviral CRISPR/Cas9 vector for HBV treatment, which may open up a new application for CRISPR therapeutics.
研究领域:
分子生物学
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纳米技术
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病毒学
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治疗学
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产品关联
DOI
高影响因子
Frontiers in Bioengineering and Biotechnology
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2022
·
IF 6.064
Yang Xiangke,Zhao Lili,Chen Qiling,Wang Nan,Shi Kan,Liu Shuwen
Organic acid metabolism by lactic acid bacteria plays a significant role in improving wine quality. During this process, the uptake of extracellular organic acids by the transporters is the first rate-limiting step. However, up to now, there is very little published research on the functional verification of organic acid transporter genes in wine lactic acid bacteria. In this study, a predicted citrate transporter gene JKL54_04345 (citP) by protein homology analysis was knocked out using a CRISPR/Cas9-based gene-editing system, and then complemented using the modified pMG36e vectors in a major wine lactic acid bacterium, Lactiplantibacillus plantarum XJ25, to verify its function in citrate metabolism for the first time. The results showed that the gene knockout mutant XJ25-ΔcitP lost the ability to utilize citric acid, while the gene complement mutant XJ25-ΔcitP-pMG36ek11-citP fully recovered the ability of citric acid utilization. Meanwhile, citP knockout and complement barely affected the utilization of l-malic acid. These indicated that citP in L. plantarum functioned as a citrate transporter and was the only gene responsible for citrate transporter. In addition, two modified plasmid vectors used for gene supplement in L. plantarum showed distinct transcription efficiency. The transcription efficiency of citP in XJ25-ΔcitP-pMG36ek11-citP mutant was 4.01 times higher than that in XJ25-ΔcitP-pMG36ek-citP mutant, and the utilization rate of citric acid in the former was 3.95 times higher than that in the latter, indicating that pMG36ek11 can be used as a high-level expression vector in lactic acid bacteria.
研究领域:
微生物学
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遗传学
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食品科学
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分子生物学
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DOI
PubMed
高影响因子
JOURNAL OF ETHNOPHARMACOLOGY
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2024
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IF 4.8
Fengyan Chen,Shiming Pu,Li Tian,Huan Zhang,Huixian Zhou,Yijing Yan,Xiaolin Hu,Qiong Wu,Xueping Chen,Shuk Han Cheng,Shisan Xu
Headings ethnopharmacological relevance Rehmanniae Radix Praeparata (RRP), a staple in traditional Chinese medicine, is derived from Rehmannia glutinosa Libosch and is renowned for its wound-healing properties. Despite its clinical prevalence, the molecular mechanisms underlying RRP's wound-healing effects have not been fully elucidated. Aim of the study This research endeavored to delineate the molecular and cellular mechanisms underlying the beneficial effects of RRP on wound healing, utilizing a zebrafish model. Materials and methods Zebrafish larvae at 3 days post-fertilization were amputated at the fin and subsequently treated with RRP. The pro-wound healing and regenerative effects of RRP were evaluated through morphological analysis, assessment of cell proliferation and apoptosis, Additionally, mechanistic insights were gained through a comprehensive approach encompassing network pharmacology analysis, cell tracing, RNA-sequencing, CRISPR/Cas9 gene editing, and pharmacological inhibition. Results Our findings demonstrate that RRP significantly accelerates caudal fin regeneration in zebrafish following injury by suppressing cell apoptosis, promoting cell proliferation, and upregulating the expression of regenerative-related genes. Furthermore, RRP triggers autophagy signals during the regenerative process, which is attenuated by the autophagy inhibitor chloroquine (CQ). Notably, the administration of RRP enhances the expression of ahr1 and ahr2 in the regenerating fin. Genetic knockout of ahr1a , ahr1b , or ahr2 using CRISPR/Cas9, or pharmacological blockade of AHR signals with the antagonist CH-223191, diminishes the regenerative potential of RRP. Remarkably, zebrafish lacking ahr2 completely lose their fin regeneration ability. Additionally, inhibition of AHR signaling suppresses autophagy signaling during fin regeneration. Conclusions This study uncovers that RRP stimulates fin regeneration in zebrafish by inducing AHR signals and, at least partially, activating the autophagy process. These findings provide novel insights into the molecular mechanisms underlying the wound-healing effects of RRP and may pave the way for the development of novel therapeutic strategies.
研究领域:
药理学
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分子生物学
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发育生物学
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中医
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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）