Cas13bt3 knockdown of VEGFA mRNA in mammalian cells and mouse retina [RNA-Seq]
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus Homo sapiens
PROVIDER: GSE259220 | GEO | 2024/03/01
REPOSITORIES: GEO
Similar Datasets
Project description:Purpose: We report expression of a new Cas13 enzyme in retinal cells and evaluate its efficacy in targeting the VEGFA mRNA to establish safety and efficacy of the enzyme got anti-VEGFA therapy. Methods: Three retinal organoids were cultured and treated, (1) Untreated, (2) AAV2.7m8.Cas13bt3.VEGFA.sgRNA, (3) AAV2.7m8.Cas13bt3.NTsgRNA. Single-cell RNA sequencing was performed to determine expression of Cas13bt3 across retinal cell types, and the corresponding VEGFA mRNA expression. Results: All retinal cell showed observable transduction and RPE cells were found most susceptible to infection, while bipolar cells had the least infection rate. Significant silencing of VEGFA was observed in RPE cells. Conclusions: AAV2.7m8 can be used for retina-wide transduction, and Cas13bt3 may be a potential new tool for control of VEGFA.
2024-03-01 | GSE259219 | GEO
Project description:The lung microvasculature is essential for gas exchange and commonly considered homogeneous. We show that Vascular endothelial growth factor A (Vegfa) from the epithelium specifies a distinct endothelial cell (EC) population in the postnatal mouse lung. Vegfa is predominantly expressed by alveolar type 1 (AT1) cells and locally required to specify a subset of ECs. Single cell RNA-seq identified 15~20% lung ECs as transcriptionally distinct and marked by Carbonic anhydrase 4 (Car4), which are specifically lost upon epithelial Vegfa deletion. Car4 ECs, unlike bulk ECs, have extensive cellular projections and are separated from AT1 cells by a limited basement membrane without any intervening pericytes. Without Car4 ECs, the alveolar space is aberrantly enlarged despite the normal appearance of myofibroblasts. Lung Car4 ECs and retina tip ECs have common and distinct transcriptional profiles. These findings support a signaling role of AT1 cells and shed light on alveologenesis.
2020-01-01 | GSE124323 | GEO
Project description:The lung microvasculature is essential for gas exchange and commonly considered homogeneous. We show that Vascular endothelial growth factor A (Vegfa) from the epithelium specifies a distinct endothelial cell (EC) population in the postnatal mouse lung. Vegfa is predominantly expressed by alveolar type 1 (AT1) cells and locally required to specify a subset of ECs. Single cell RNA-seq identified 15~20% lung ECs as transcriptionally distinct and marked by Carbonic anhydrase 4 (Car4), which are specifically lost upon epithelial Vegfa deletion. Car4 ECs, unlike bulk ECs, have extensive cellular projections and are separated from AT1 cells by a limited basement membrane without any intervening pericytes. Without Car4 ECs, the alveolar space is aberrantly enlarged despite the normal appearance of myofibroblasts. Lung Car4 ECs and retina tip ECs have common and distinct transcriptional profiles. These findings support a signaling role of AT1 cells and shed light on alveologenesis.
2020-01-01 | GSE124324 | GEO
Project description:Current gene silencing tools based on RNA interference (RNAi) or more recently the CRISPR-Cas13 systems have critical drawbacks in such as off-target effects (RNAi) or collateral mRNA cleavage (CRISPR-Cas13). Thus, a more specific method of gene knockdown has been demanded. Here we developed “CRISPRδ”, an approach for translational silencing, harnessing the catalytically inactive Cas13 proteins (dCas13). Owing to the tight association with mRNA, dCas13 serves as a physical roadblock for scanning ribosomes in translation initiation, but not for elongating ribosomes, with no effect on mRNA stability. Guide RNAs covering the start codon lead to the highest efficacy, irrespective of translation initiation mechanisms: cap-dependent, IRES-dependent, or RAN translations. Strikingly, genome-wide ribosome profiling revealed extremely high specificity of CRISPRδ in a gene knockdown. Moreover, the fusion of a translation repressor to dCas13 ensured further improvement of the knockdown efficacy. Our method provides a framework for translational repression-based gene silencing in eukaryotes.
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Project description:VEGFA signaling is crucial for physiological and pathological angiogenesis and hematopoiesis. Although many context-dependent signaling pathways downstream of VegfA have been uncovered, vegfa transcriptional regulation in vivo is unclear. Here, we show that ETS transcription factor Etv6 positively regulates vegfa expression during blood stem cell development through multiple transcriptional inputs. In agreement with its established repressive functions, Etv6 directly inhibits expression of vegfa repressor foxo3. Surprisingly, it also directly activates expression of vegfa activator klf4. Finally, it indirectly binds to vegfa promoter by the recruiment of Klf4. Thus, our work uncovers a dual function for Etv6, as both a transcriptional repressor and activator, in controlling a major signaling pathway.
2019-01-16 | GSE115224 | GEO
Project description:VEGFA signaling is crucial for physiological and pathological angiogenesis and hematopoiesis. Although many context-dependent signaling pathways downstream of VegfA have been uncovered, vegfa transcriptional regulation in vivo is unclear. Here, we show that ETS transcription factor Etv6 positively regulates vegfa expression during blood stem cell development through multiple transcriptional inputs. In agreement with its established repressive functions, Etv6 directly inhibits expression of vegfa repressor foxo3. Surprisingly, it also directly activates expression of vegfa activator klf4. Finally, it indirectly binds to vegfa promoter by the recruiment of Klf4. Thus, our work uncovers a dual function for Etv6, as both a transcriptional repressor and activator, in controlling a major signaling pathway.
2019-01-16 | GSE115220 | GEO
Project description:The bivalent domain at promoter region is a unique epigenetic feature poised for activation or repression during cell differentiation in embryonic stem cell. However, the function of bivalent domains in already differentiated cells remains exclusive. By profiling the epigenetic landscape of endothelial cells during VEGFA stimulation, we discovered that bivalent domains are widespread in endothelial cells and preferentially marked genes responsive to VEGFA. The bivalent domains responsive to VEGFA have more permissive chromatin environment comparing to other bivalent domains. The initial activation of bivalent genes depends on RNAPII pausing release induced by EZH1 rather than removal of H3K27me3. The later suppression of bivalent gene expression depended on KDM5A recruitment by its interaction with PRC2. Importantly, EZH1 promoted both in vitro and in vivo angiogenesis by upregulating EGR3, whereas KDM5A dampened angiogenesis. Collectively, this study demonstrated a novel dual function of bivalent domains in endothelial cells to control VEGF responsiveness and angiogenesis.
2020-10-17 | GSE139077 | GEO
Project description:Epithelial Vegfa specifies a distinct endothelial population in the mouse lung
| PRJNA511606 | ENA
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