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CRISPR/Cas9 genome editing demonstrates that CCM3 modulates endothelial miRNA expression

ABSTRACT: CRISPR/Cas9 genome editing demonstrates that CCM3 modulates endothelial miRNA expression

PROVIDER: PRJNA473162 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR7221070.fastq.gz	Fastqsanger.gz
	SRR7221063.fastq.gz	Fastqsanger.gz
	SRR7221064.fastq.gz	Fastqsanger.gz
	SRR7221065.fastq.gz	Fastqsanger.gz
	SRR7221066.fastq.gz	Fastqsanger.gz

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CRISPR/Cas9 genome editing demonstrates that CCM3 modulates endothelial miRNA expression

Project description:CCM3 regulates blood-brain-barrier integrity and vascular maturation in vivo. CCM3 loss-of-function variants predispose to cerebral cavernous malformations (CCM). Various signalling pathways are deregulated upon CCM3 depletion in endothelial cells (ECs). In this study, we established a crRNA:tracrRNA:Cas9 RNP approach to efficiently knockout CCM3 in human ECs and studied the molecular and functional effects of its long-term inactivation. Using small RNA sequencing, we show that CCM3 regulates the expression of aging‑associated miRNAs.

2019-01-18 | GSE114947 | GEO

mRNA expression changes in CCM3-deficient endothelial cells

Project description:Loss-of-function variants in CCM3/PDCD10 predispose to cerebral cavernous malformations (CCMs) that are vascular lesion of the central nervous system. Using CRISPR/Cas9 genome editing and RNA sequencing, we have shown that long-term inactivation of CCM3 in human endothelial cells dysregulates fibronectin expression and thus impairs the assembly of a functional fibronectin matrix by endothelial cells.

2020-07-06 | GSE137425 | GEO

The fibronectin mediated endothelial rescue effect in CCM3-deficient cells is independent from acute mRNA changes

Project description:Using CRISPR/Cas9 genome editing and RNA sequencing, we demonstrate that fibronectin supplementation restores the impaired spheroid formation and attenuates actin stress fiber assembly in CCM3-/- endothelial cells independently of acute gene expression differences. In particular, there was no significant downregulation of well-known key players in CCM pathogenesis like KLF2 or KLF4.

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Targeted DNA-seq of Human HepG2 cells following editing with CRISPR/Cas9

Project description:We performed a large-scale genome-wide characterisation of indels generated following editing with CRISPR/Cas9. We used pools of sgRNAs and performed targeted capture and sequencing of the edited regions in HepG2 cells.

2018-12-13 | E-MTAB-7095 | biostudies-arrayexpress

NGS data of Nicotiana benthamiana 16c plants after CRISPR/Cas9 gene editing

Project description:Provided data came from a detailed study on Nicotiana benthamiana 16c plants where we use Tobacco Rattle Virus (TRV) as a molecular switch to change the chromatin state of a reporter gene (P35S::GFP) from an actively transcribed to a transcriptionally silenced state. Our approach enables us to interrogate different chromatin states of the same locus with the same set of CRISPR/Cas9 genome editing reagents and systematically describe the effect of chromatin state on the frequency and type of mutations induced at various Cas9 targets in a huge set of independently edited cells.

2022-05-04 | E-MTAB-10256 | biostudies-arrayexpress

MiSeq paired-end sequencing of HEK293T cells stably expressing a Cas9/gRNA-dependent editable reporter vector to test novel nucleobase-editing techniques

Project description:The goal of these experiments were to test the on-target and target-adjacent editing efficiencies of different single-nucleobase editing systems. Previous studies have shown that tethering DNA mutating enzymes to Cas9-nickase-UGI complexes results in editing of chromosomal DNA. However, these editing events encompass undesirable target-adjacent nucleobase edits. Here, we characterize a novel approach that reduces the frequency of target-adjacent editing while maintaining a high level of on-target editing.

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Increase genome editing efficiency by Cas9 transcriptional activator and histone acetyltransferase activator

Project description:The CRISPR/Cas9 system shows diverse levels of genome editing activities on eukaryotic genomic DNA targets, and experiments desire high-efficiency targets. Here we show that chromatin open status is a pivotal determinant of the Cas9 editing activity in mammalian cells, and increasing chromatin accessibility can efficiently improve Cas9 genome editing activity. However, the strategy that fusing the VP64 transcriptional activation domain at the C-terminus of Cas9 can only promote genome editing activity slightly at most tested CRISPR/Cas9 targets in Lenti-X 293T cells. Because histone acetylation increases eukaryotic chromatin accessibility, we further improve genome editing by elevating histone acetylation. We demonstrate that promoting histone acetylation using histone acetyltransferase (HAT) activator YF-2 can improve genome editing from Cas9 and more robustly from the Cas9 transcriptional activator. This provides a strategy to improve CRISPR/Cas9 genome editing activity and enables broader gRNA target choices in eukaryotes.

2022-04-21 | GSE200943 | GEO

Determinants for efficient editing with Cas9-mediated recombineering in Escherichia coli

Project description:In E. coli, editing efficiency (EE) with Cas9-mediated recombineering varies across targets due to differences in the level of Cas9:gRNA DNA double-strand break (DSB)-induced cell death. We found that EE with the same gRNA and repair template can also change with target position, cas9 promoter strength, and growth conditions. Incomplete editing, off-target activity, non-targeted mutations, and failure to cleave target DNA even if Cas9 is bound also compromise EE. These effects on EE were gRNA-specific. We propose that differences in the efficiency of Cas9:gRNA-mediated DNA DSBs and differences in rates of dissociation of Cas9:gRNA complexes from target sites account for the observed variations in EE between gRNAs. We show that editing behavior using the same gRNA can be modified by mutating the gRNA spacer, which changes the DNA DSB activity. Finally, we discuss how variable editing with different gRNAs could limit high-throughput applications and provide strategies to overcome these limitations.

2020-06-06 | GSE132139 | GEO

Genome-wide specificity of genome editing by both CRISPR-Cas9 and TALEN in the model plant Physcomitrium patens

Project description:CRISPRs and TALENs are efficient systems for gene editing in many organisms including plants. In many cases the CRISPR-Cas or TALEN modules are expressed in the plant cell only transiently. Theoretically, transient expression of the editing modules should limit unexpected effects compared to stable transformation. However, very few studies have measured the off-target and unpredicted effects of editing strategies on the plant genome, and none of them have compared these two major editing systems. We conducted a comprehensive genome-wide investigation of off-target mutations using either a CRISPR-Cas9 or a TALEN strategy. We observed a similar number of SNVs and InDels for the two editing strategies compared to control non-transfected plants, with an average of 8.25 SNVs and 19.5 InDels for the CRISPR-edited plants, and an average of 17.5 SNVs and 32 InDels for the TALEN-edited plants. Interestingly, a comparable number of SNVs and InDels could be detected in the PEG-treated control plants. This shows that except for the on-target modifications, the gene editing tools used in this study did not show a significant off-target activity nor unpredicted effects on the genome, and that the PEG treatment in itself was probably the main source of mutations found in the edited plants.

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Shortening the half-life of Cas9 maintains its gene editing ability and reduces neuronal toxicity

Project description:Virus-mediated expression of CRISPR/Cas9 has been actively used for genome editing in animal models of genetic disorders, including neurological diseases, but the consequences of overexpressing bacterial Cas9 in the mammalian brain remain unknown. Through RNA-seq analysis, we found that virus-mediated expression of Cas9 caused systematic changes in genes involved in neuronal functions. We also generated a short-lived version of Cas9, which maintains its genome editing capacity, but significantly alleviates neurotoxicity caused by overexpressed Cas9. Thus, modification of Cas9 by shortening its half-life would help develop CRISPR/Cas9-based therapeutic approaches for treating genetic neurological disorders.

2018-12-04 | GSE122072 | GEO

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