Nanoscale
·
2017
·Vol. 10
·期 3
·1063-1071
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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
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遗传学
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纳米医学
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PubMed
高影响因子
NUCLEIC ACIDS RESEARCH
·
2025
·Vol. 53
·期 6
·
IF 13.1
Mao Shiqi,Wu Ruonan,Luo Weibang,Qin Jinshan,Chen Antony K
Clustered regularly interspaced short palindromic repeats (CRISPR)-based imaging tools that utilize fluorescently tagged single-guide RNAs (sgRNAs) have enabled versatile analysis of the dynamics of single genomic loci, but the accuracy may be hindered by nonspecific subnuclear probe accumulation, generating false-positive foci in cell nuclei. By examining the subcellular localizations of sgRNA expression plasmids, their RNA transcripts, and several RNA-binding proteins, we found that spuriously transcribed (cryptic) transcripts, produced by sgRNA expression plasmids, are the major contributors of false-positive signals, independent of sgRNA scaffold design or effector probe (i.e. RNA aptamer- or oligonucleotide-based probes) used. These transcripts interact with the paraspeckle core proteins, but not with the sgRNA expression plasmids or the paraspeckle RNA scaffold NEAT1_2, to form nuclear bodies that display liquid-like properties including sphericality, fusion competence, and sensitivity to 1,6-hexanediol. Transfecting sgRNA transcription units (i.e. sgRNA expression cassettes), lacking the plasmid backbones, reduces false-positive signals and enhances genomic imaging accuracy. Overall, this study unveils previously undescribed activities of cryptic plasmid transcripts and presents an easy-to-adapt strategy that can potentially improve the precision of CRISPR-based imaging systems that implement fluorescently tagged sgRNAs.
研究领域:
分子生物学
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遗传学
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生物物理学
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细胞生物学
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PubMed
高影响因子
JOURNAL OF NANOBIOTECHNOLOGY
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2024
·Vol. 22
·期 1
·1-29
·
IF 10.6
Yu Xiao-Jun,Zhao Yuan-Ting,Abudouaini Haimiti,Zou Peng,Li Tian-Qi,Bai Xiao-Fan,Wang Shan-Xi,Guan Jian-Bin,Li Meng-wei,Wang Xiao-dong,Wang Ying-guang,Hao Ding-Jun
Methods Single-cell transcriptomics and high-throughput transcriptomics were used to screen factors significantly correlated with intervertebral disc degeneration (IDD). Expression changes of CFIm25 were determined via RT-qPCR and Western blot. NP cells were isolated from mouse intervertebral discs and induced to degrade with TNF-α and IL-1β. CFIm25 was knocked out using CRISPR-Cas9, and CFIm25 knockout and overexpressing nucleus pulposus (NP) cell lines were generated through lentiviral transfection. Proteoglycan expression, protein expression, inflammatory factor expression, cell viability, proliferation, migration, gene expression, and protein expression were analyzed using various assays (alcian blue staining, immunofluorescence, ELISA, CCK-8, EDU labeling, transwell migration, scratch assay, RT-qPCR, Western blot). The GelMA-HAMA hydrogel loaded with APET×2 polypeptide and sgRNA was designed, and its effects on NP regeneration were assessed through in vitro and mouse model experiments. The progression of IDD in mice was evaluated using X-ray, H&E staining, and Safranin O-Fast Green staining. Immunohistochemistry was performed to determine protein expression in NP tissue. Proteomic analysis combined with in vitro and in vivo experiments was conducted to elucidate the mechanisms of hydrogel action. Results CFIm25 was upregulated in IDD NP tissue and significantly correlated with disease progression. Inhibition of CFIm25 improved NP cell degeneration, enhanced cell proliferation, and migration. The hydrogel effectively knocked down CFIm25 expression, improved NP cell degeneration, promoted cell proliferation and migration, and mitigated IDD progression in a mouse model. The hydrogel inhibited inflammatory factor expression (IL-6, iNOS, IL-1β, TNF-α) by targeting the p38/NF-κB signaling pathway, increased collagen COLII and proteoglycan Aggrecan expression, and suppressed NP degeneration-related factors (COX-2, MMP-3). Conclusion The study highlighted the crucial role of CFIm25 in IDD and introduced a promising therapeutic strategy using a porous spherical GelMA-HAMA hydrogel loaded with APET×2 polypeptide and sgRNA. This innovative approach offers new possibilities for treating degenerated intervertebral discs. Graphical Abstract Molecular mechanism of GCA targeting CFIm25 in NP cells to promote regeneration of degenerated intervertebral disc NP
研究领域:
生物医学工程
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再生医学
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组织工程
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分子生物学
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细胞生物学
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骨科
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PubMed
高影响因子
VETERINARY MICROBIOLOGY
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2024
·
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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PubMed
高影响因子
MICROCHEMICAL JOURNAL
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2026
·
IF 5.2
Shuo Zhang,Ting Yang,Rui Lu,Huarong Pang,Huakang Li,Bin Li,Yuzhe Song
Cb Ago selectively depletes wild-type TP53, eliminating background interference for mutant analysis. • Cas9 nickase generates ssDNA initiators to trigger EXPAR under isothermal conditions. • Amplified products disrupt G-quadruplex/ThT fluorescence, enabling a signal-off readout. • The assay achieves 0.14 fM detection limit and single-nucleotide resolution. • Validated in serum and cells; consistent with PCR; discriminates cancer from normal cells. Accurate detection of rare mutant TP53 in the presence of abundant wild-type background remains a major challenge for early diagnosis of colorectal cancer. Here, we report a background-reduced analytical strategy that integrates Cb Ago-mediated wild-type depletion with Cas9 nickase (Cas9n)/sgRNA-triggered exponential amplification reaction (EXPAR) for highly sensitive and specific mutant TP53 analysis. Cb Ago programmed with a guide DNA selectively cleaves wild-type TP53 while preserving the mutant target, thereby eliminating interference from the overwhelming wild-type background. The remaining mutant TP53 is then recognized by two Cas9n/sgRNA complexes, which generate single-stranded initiator fragments through polymerase-assisted strand displacement. These initiators trigger an EXPAR cascade that ultimately disrupts the G-quadruplex structure of a thioflavin T (ThT) fluorescent probe, producing a signal-off readout. The method achieves a detection limit as low as 0.14 fM with excellent single-nucleotide discrimination capability. Its practical utility is validated through recovery experiments, differentiation of cancer cells from normal cells, and high consistency with PCR-based methods in clinical tissue samples. This Cb Ago-Cas9n-EXPAR platform offers a robust and isothermal tool for early diagnosis of TP53-mutated colorectal cancer and can be readily reprogrammed for other clinically relevant single-nucleotide variants. Download: Download high-res image (123KB) Download: Download full-size image
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FREE RADICAL RESEARCH
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2024
·
IF 3.6
Xue Peng,Xi Tan,Li Dai,Wei Xia,Zhao Wu
This research investigates the interplay between Reactive Oxygen Species (ROS) and Apelin (APLN) in regulating autophagy, with implications for placental cell senescence and apoptosis in pre-eclampsia (PE). We manipulated APLN expression using sgRNA to study its effects on ROS levels and subsequent cellular responses. Our findings reveal that APLN overexpression elevates ROS production, accelerating cellular senescence and apoptosis. In contrast, silencing APLN enhances autophagy, thereby diminishing cellular aging and apoptosis. These outcomes were confirmed in vitro and in vivo experiments, establishing a causative relationship between ROS-mediated APLN modulation and altered placental cell dynamics in PE. The results suggest potential therapeutic targets within the ROS and APLN pathways to alleviate detrimental changes in the placenta, offering new strategies for the clinical management of PE. This study emphasizes the crucial role of autophagy in placental health and sets the stage for future investigations into therapeutic interventions for pregnancy-related complications.
研究领域:
妇产科学
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分子生物学
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细胞生物学
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药理学
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PubMed
高影响因子
BIOTECHNOLOGY LETTERS
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2023
·Vol. 45
·期 3
·401-410
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IF 2.7
Tian Li,Yan Bingyu,Huo Dandan,Sun Wenhui,Cui Sufang,Li Xiaojing,Zhang Xiangmei,Dong Huijun
Objectives To develop a modified CRISPR/Cas9 system with the β-glucuronidase (GusA) reporter and a dual sgRNA cassette for Nonomuraea gerenzanensis ( N. gerenzanensis ). Results With the aid of a visual GusA reporter, the complicated and tedious process of cloning and gene identification could be abandoned entirely in the genetic editing of N. gerenzanensis . Moreover, introducing a dual sgRNA cassette into the CRISPR/Cas9 system significantly improved gene deletion efficiency compared to the single sgRNA element. Furthermore, the length of the homologous flanking sequences set to the lowest value of 500 bp in this system could still reach the relatively higher conjugation transfer frequency. Conclusions The enhanced CRISPR/Cas9 system could efficiently perform genetic manipulation on the rare actinomycete N. gerenzanensis .
研究领域:
微生物学
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分子生物学
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遗传工程
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PubMed
高影响因子
WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY
·
2024
·Vol. 40
·期 6
·1-13
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IF 4
Zhao Siji,Yin Ruya,Zhang Mengwei,Zhai Ziqi,Shen Zhen,Mou Yan,Xu Dan,Zhou Ligang,Lai Daowan
The endophytic fungus Berkleasmium sp. Dzf12 that was isolated from Dioscorea zingiberensis , is a proficient producer of palmarumycins, which are intriguing polyketides of the spirobisnaphthalene class. These compounds displayed a wide range of bioactivities, including antibacterial, antifungal, and cytotoxic activities. However, conventional genetic manipulation of Berkleasmium sp. Dzf12 is difficult and inefficient, partially due to the slow-growing, non-sporulating, and highly pigmented behavior of this fungus. Herein, we developed a CRISPR/Cas9 system suitable for gene editing in Berkleasmium sp. Dzf12. The protoplast preparation was optimized, and the expression of Cas9 in Berkleasmium sp. Dzf12 was validated. To assess the gene disruption efficiency, a putative 1, 3, 6, 8-tetrahydroxynaphthalene synthase encoding gene, bdpks , involved in 1,8-dihydroxynaphthalene (DHN)-melanin biosynthesis, was selected as the target for gene disruption. Various endogenous sgRNA promoters were tested, and different strategies to express sgRNA were compared, resulting in the construction of an optimal system using the U6 snRNA-1 promoter as the sgRNA promoter. Successful disruption of bdpks led to a complete abolishment of the production of spirobisnaphthalenes and melanin. This work establishes a useful gene targeting disruption system for exploration of gene functions in Berkleasmium sp. Dzf12, and also provides an example for developing an efficient CRISPR/Cas9 system to the fungi that are difficult to manipulate using conventional genetic tools.
研究领域:
分子生物学
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真菌遗传学
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生物技术
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植物化学
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PubMed
高影响因子
Frontiers in Cell and Developmental Biology
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2022
·
IF 6.081
Deng Changping,Hu Fabiao,Zhao Zhangting,Zhou Yiwen,Liu Yuping,Zhang Tong,Li Shihui,Zheng Wenyun,Zhang Wenliang,Wang Tianwen,Ma Xingyuan
Quantitative analysis and regulating gene expression in cancer cells is an innovative method to study key genes in tumors, which conduces to analyze the biological function of the specific gene. In this study, we found the expression levels of Survivin protein (BIRC5) and P-glycoprotein (MDR1) in MCF-7/doxorubicin (DOX) cells (drug-resistant cells) were significantly higher than MCF-7 cells (wild-type cells). In order to explore the specific functions of BIRC5 gene in multi-drug resistance (MDR), a CRISPR/Cas9-mediated knocking-in tetracycline (Tet)-off regulatory system cell line was established, which enabled us to regulate the expression levels of Survivin quantitatively (clone 8 named MCF-7/Survivin was selected for further studies). Subsequently, the determination results of doxycycline-induced DOX efflux in MCF-7/Survivin cells implied that Survivin expression level was opposite to DOX accumulation in the cells. For example, when Survivin expression was down-regulated, DOX accumulation inside the MCF-7/Survivin cells was up-regulated, inducing strong apoptosis of cells (reversal index 118.07) by weakening the release of intracellular drug from MCF-7/Survivin cells. Also, down-regulation of Survivin resulted in reduced phosphorylation of PI3K, Akt, and mTOR in MCF-7/Survivin cells and significantly decreased P-gp expression. Previous studies had shown that PI3K/Akt/mTOR could regulate P-gp expression. Therefore, we speculated that Survivin might affect the expression of P-gp through PI3K/Akt/mTOR pathway. In summary, this quantitative method is not only valuable for studying the gene itself, but also can better analyze the biological phenomena related to it.
研究领域:
癌症生物学
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分子生物学
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基因工程
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药理学
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PubMed
高影响因子
BIOMACROMOLECULES
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2022
·
IF 6.978
Zhiyu Yang,Lin Lin,Zhaopei Guo,Xiaoya Guo,Zhaohui Tang,Huayu Tian,Xuesi Chen
The relatively low transfection efficiency limits further application of polymeric gene carriers. It is imperative to exploit a universal and simple strategy to enhance the gene transfection efficiency of polymeric gene carriers. Herein, we prepared a cationic polypeptide poly(γ-aminoethylthiopropyl-l-glutamate) (PALG-MEA, termed PM) with a stable α-helical conformation, which can significantly improve the gene transfection efficiency of cationic polymers. PM can be integrated into polymeric gene delivery systems noncovalently through electrostatic interactions. With the assistance of PM, polymeric gene delivery systems exhibited excellent cellular uptake and endosomal escape, thereby enhancing transfection efficiency. The transfection enhancement effect of PM was applicable to a variety of cationic polymers such as polyethylenimine (PEI), poly-l-lysine (PLL), and polyamidoamine (PAMAM). The ternary gene delivery system PM/pshVEGF/PEI exhibited an excellent antitumor effect against the B16F10 tumor model. Moreover, we demonstrated that PM could also enhance the delivery of gene editing systems (sgRNA-Cas9 plasmids). This work provides a facile and effective strategy for constructing polymeric gene delivery systems with a high transfection efficiency.
研究领域:
遗传学
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生物医学工程
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分子生物学
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基因治疗
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PubMed
高影响因子
Acta Pharmaceutica Sinica B
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2022
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IF 14.903
Zekun Li,Yongchun Pan,Shiyu Du,Yayao Li,Chao Chen,Hongxiu Song,Yueyao Wu,Xiaowei Luan,Qin Xu,Xiaoxiang Guan,Yujun Song,Xin Han
Near-infrared (NIR)-light-triggered nanomedicine, including photodynamic therapy (PDT) and photothermal therapy (PTT), is growing an attractive approach for cancer therapy due to its high spatiotemporal controllability and minimal invasion, but the tumor eradication is limited by the intrinsic anti-stress response of tumor cells. Herein, we fabricate a tumor-microenvironment responsive CRISPR nanoplatform based on oxygen-deficient titania (TiO 2- x ) for mild NIR-phototherapy. In tumor microenvironment, the overexpressed hyaluronidase (HAase) and glutathione (GSH) can readily destroy hyaluronic acid (HA) and disulfide bond and releases the Cas9/sgRNA from TiO 2- x to target the stress alleviating regulators, i.e. , nuclear factor E2-related factor 2 (NRF2) and heat shock protein 90 α (HSP90 α ), thereby reducing the stress tolerance of tumor cells. Under subsequent NIR light illumination, the TiO 2- x demonstrates a higher anticancer effect both in vitro and in vivo . This strategy not only provides a promising modality to kills cancer cells in a minimal side-effects manner by interrupting anti-stress pathways but also proposes a general approach to achieve controllable gene editing in tumor region without unwanted genetic mutation in normal environments.
研究领域:
癌症治疗
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纳米技术
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基因编辑
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光疗
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生物材料
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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）