- 标题
- 摘要
- 关键词
- 实验方案
- 产品
-
Near-infrared upconversion–activated CRISPR-Cas9 system: A remote-controlled gene editing platform
摘要： 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.
关键词： cancer therapeutics,upconversion nanoparticles,gene editing,CRISPR-Cas9,near-infrared
更新于2025-11-21 11:08:12
-
Functional Assessment of Patient-Derived Retinal Pigment Epithelial Cells Edited by CRISPR/Cas9
摘要： Retinitis pigmentosa is the most common form of inherited blindness and can be caused by a multitude of different genetic mutations that lead to similar phenotypes. Specifically, mutations in ubiquitously expressed splicing factor proteins are known to cause an autosomal dominant form of the disease, but the retina-specific pathology of these mutations is not well understood. Fibroblasts from a patient with splicing factor retinitis pigmentosa caused by a missense mutation in the PRPF8 splicing factor were used to produce three diseased and three CRISPR/Cas9-corrected induced pluripotent stem cell (iPSC) clones. We differentiated each of these clones into retinal pigment epithelial (RPE) cells via directed differentiation and analyzed the RPE cells in terms of gene and protein expression, apicobasal polarity, and phagocytic ability. We demonstrate that RPE cells can be produced from patient-derived and corrected cells and they exhibit morphology and functionality similar but not identical to wild-type RPE cells in vitro. Functionally, the RPE cells were able to establish apicobasal polarity and phagocytose photoreceptor outer segments at the same capacity as wild-type cells. These data suggest that patient-derived iPSCs, both diseased and corrected, are able to differentiate into RPE cells with a near normal phenotype and without differences in phagocytosis, a result that differs from previous mouse models. These RPE cells can now be studied to establish a disease-in-a-dish system relevant to retinitis pigmentosa.
关键词： retinal pigment epithelial cells,CRISPR/Cas9,retinitis pigmentosa,PRPF8,induced pluripotent stem cells
更新于2025-09-23 15:23:52
-
Manipulating Living Systems by Light; 生命現象の光操作技術の創出;
摘要： Complex gene networks are essential for diverse biological phenomena, such as cellular programming, metabolism, homeostasis, memory formation, and circadian rhythm. To understand these biological phenomena, including diseases, and to utilize or modify them, approaches that enable optical control of the genome are required. We developed new tools for targeted gene manipulation based on optical control of the CRISPR-Cas9 system and Cre-loxP system. These tools could greatly facilitate understanding of a variety of gene functions and prove useful in biomedical applications. Genome engineering technology and optogenetics technology have emerged as different technologies from each other so far. Our studies merge these emerging research fields together.
关键词： optogenetics,photoswitching protein,cell differentiation,Cre-loxP system,genome editing,CRISPR-Cas9 system
更新于2025-09-23 15:22:29
-
Rapid identification of human mast cell degranulation regulators using functional genomics coupled to high-resolution confocal microscopy
摘要： Targeted functional genomics represents a powerful approach for studying gene function in vivo and in vitro. However, its application to gene expression studies in human mast cells has been hampered by low yields of human mast cell cultures and their poor transfection efficiency. We developed an imaging system in which mast cell degranulation can be visualized in single cells subjected to shRNA knockdown or CRISPR–Cas9 gene editing. By using high-resolution confocal microscopy and a fluorochrome-labeled avidin probe, one can directly assess the alteration of functional responses, i.e., degranulation, in single human mast cells (10–12 weeks old). The elimination of a drug or marker selection step avoids the use of potentially toxic treatment procedures, and the brief hands-on time of the functional analysis step enables high-throughput screening of shRNA or CRISPR–Cas9 constructs to identify genes that regulate human mast cell degranulation. The ability to analyze single cells substantially reduces the total number of cells required and enables the parallel visualization of the degranulation profiles of both edited and non-edited mast cells, offering a consistent internal control not found in other protocols. Moreover, our protocol offers a flexible choice between RNA interference (RNAi) and CRISPR–Cas9 genome editing for perturbation of gene expression using our human mast cell single-cell imaging system. Perturbation of gene expression, acquisition of microscopy data and image analysis can be completed within 5 d, requiring only standard laboratory equipment and expertise.
关键词： confocal microscopy,human mast cells,CRISPR–Cas9,degranulation,shRNA,functional genomics
更新于2025-09-23 15:19:57
-
A Simple and Efficient Method for Designing Broadband Terahertz Absorber Based on Singular Graphene Metasurface
摘要： In the present study, Amh was found to be abundantly expressed in the granulosa cells of the primary growth follicles, and Amhr2 in the granulosa cells, oogonia and phase I oocytes in tilapia by immunohistochemistry. In addition, Amh and Amhr2 were also found to be expressed in the brain and pituitary. Heterozygous mutation of either amh or amhr2 resulted in increased primary growth follicles and decreased fertility, and homozygous mutation resulted in hypertrophic ovaries with significantly increased primary follicles and failed transition from primary to vitellogenic follicles. Expression of gnrh3 in the brain, fsh and lh in the pituitary and serum E2 concentration were significantly decreased in both mutants. Significantly increased apoptosis of follicle cells was observed in both mutants. However, administration of E2 failed to rescue the folliculogenesis defects of the mutants. Our results suggested that Amh acts in a dose-dependent manner by binding Amhr2 in tilapia.
关键词： Ovary hypertrophy,CRISPR/Cas9,Anti-Müllerian hormone,Fertility,Folliculogenesis arrest
更新于2025-09-12 10:27:22
-
Targeted deletion of an NRL- and CRX-regulated alternative promoter specifically silences FERM and PDZ domain containing 1 ( <i>Frmpd1</i> ) in rod photoreceptors
摘要： Regulation of cell type-specific gene expression is critical for generating neuronal diversity. Transcriptome analyses have unraveled extensive heterogeneity of transcribed sequences in retinal photoreceptors because of alternate splicing and/or promoter usage. Here we show that Frmpd1 (FERM and PDZ domain containing 1) is transcribed from an alternative promoter specifically in the retina. Electroporation of Frmpd1 promoter region, -505 to +382 bp, activated reporter gene expression in mouse retina in vivo. A proximal promoter sequence (-8 to +33 bp) of Frmpd1 binds to NRL and CRX, two rod-specific differentiation factors, and is necessary for activating reporter gene expression in vitro and in vivo. CRISPR/Cas9-mediated deletion of the genomic region including NRL and CRX binding sites in vivo completely eliminated Frmpd1 expression in rods and dramatically reduced expression in rod bipolar cells, thereby overcoming embryonic lethality caused by germline Frmpd1 deletion. Our studies demonstrate that a cell-type-specific regulatory control region is a credible target for creating loss-of-function alleles of widely-expressed genes.
关键词： rod photoreceptors,Frmpd1,NRL,CRX,alternative promoter,CRISPR/Cas9
更新于2025-09-10 09:29:36
-
Concurrent live imaging of DNA double-strand break repair and cell-cycle progression by CRISPR/Cas9-mediated knock-in of a tricistronic vector
摘要： Cell-cycle progression can be arrested by ionizing radiation-induced DNA double-strand breaks (DSBs). Although DSBs are patched by DSB repair systems, which comprise proteins such as p53-binding protein 1 (53BP1), the relationship between DSB repair progression and cell-cycle status in living cells is unclear. The probe FUCCI (fluorescent ubiquitination-based cell-cycle indicator) was previously developed for visualizing cell-cycle status. Here, we established novel live-imaging probes based on custom-designed plasmids designated “Focicles” harboring a tricistronic compartment encoding distinct fluorescent proteins ligated to the murine 53BP1 foci-forming region (FFR) and two cell-cycle indicators that are known components of FUCCI (hCdt1 and hGmnn). We used CRISPR/Cas9-mediated genome editing to obtain Focicle knock-in cell lines in NIH3T3 cells, which were subject to X-ray irradiation that induced comparable numbers of Focicle and endogenous-53BP1 foci. In addition, the Focicle probes enabled the kinetic analysis of both DSB repair and cell-cycle arrest/progression after irradiation, demonstrating that the Focicle knock-in cells progressed to cell division after DNA damage elimination. These newly developed probes can help to gain a better understanding of the dynamics of DSB repair and cell-cycle control to in turn guide cancer treatment development and cancer-risk assessments.
关键词： FUCCI,CRISPR/Cas9,53BP1,DSB repair,live imaging,cell-cycle progression,DNA double-strand breaks
更新于2025-09-10 09:29:36