Ontology highlight
ABSTRACT:
PROVIDER: PRJNA667456 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR12774500.fastq.gz | Fastqsanger.gz | |||
| SRR12774501.fastq.gz | Fastqsanger.gz | |||
| SRR12774502.fastq.gz | Fastqsanger.gz | |||
| SRR12774503.fastq.gz | Fastqsanger.gz | |||
| SRR12774504.fastq.gz | Fastqsanger.gz |
Items per page: 5 1 - 5 of 936 |
Similar Datasets
Project description:Precise Genome Editing in vivo with Single, Canonical AAV Vectors. Encoding Guide RNA, Cas9, and Homologous Repair Donor
| PRJNA668612 | ENA
Project description:Since LAMC2 is a secreted molecule present in the extracellular matrix of the cells, was designed a strategy based on CRISPR/Cas9-mediated homologous recombination to mark LAMC2 cells in human PDACs. A Cas9 single-guide RNAs complementary to sequences overlapping the stop codon of the LAMC2 locus was designed and a donor vector that contained LAMC2 homology arms flanking an EGFP reporter cassette positioned immediately upstream of the stop codon was generated. LF2A self-cleavage peptide in frame with EGFP so that LAMC2-EGFP locus was expressed as a single mRNA was added, whereas the resulting polypeptide was cleaved in the two encoded proteins, LAMC2 and EGFP. L3.6pl and PANC-1 cells were nucleofected with the donor vector together with a guide-RNA-Cas9 (guide). The engineered cells were subcutaneously injected in CD1 male mice and RNA-seq was performed on LAMC2-EGFP+ and EGFP- -derived tumors.
2022-04-11 | E-MTAB-11597 | biostudies-arrayexpress
Project description:Mouse retinas and livers were injected with AAV2/8 vectors encoding for SpCas9 under control of tissue-specific promoters, as well as a donor DNA and gRNA used to perform targeted integration in the two target loci (rhodopsin for the retina and albumin for the liver) via non-homologous end joining. Retinas and livers were harvested one month after injection and DNA was extracted. PCR products comprising the 5' and 3' junctions between the endogenous loci and the inserted donor DNA were amplified and used for library generation. NGS analysis allowed characterization of the kinds and relative frequency of insertions and deletions generated at the junction points in order to better understand the precision of integration achieved by this gene editing approach.
2022-01-13 | GSE158771 | GEO
Project description:We previously found that an adeno-associated virus (AAV) vector containing S. aureus Cas9 and a multi-target guide (g)RNA could integrate into the genome and prematurely terminate transcription of Ube3a-ATS, a long non-coding RNA. Here, we assessed the performance of three additional AAV vectors containing S. aureus Cas9 and twenty-five vectors containing N. meningococcus Cas9, all targeting single sites within Ube3a-ATS. We found that none of these single-target gRNA vectors were as effective as multi-target gRNA vectors at reducing Ube3a-ATS expression in neurons. We also developed a new anchored multiplex PCR sequencing (AMP-seq) method and analysis pipeline to quantify the relative frequency of all possible editing events at target sites, including unresolved double-stranded breaks (DSBs) and capture of foreign DNA. We found that integration of AAV was the most frequent editing event (67-89% of all edits) at three different single target sites, far surpassing insertions and deletions (indels). None of the most frequently observed indels was capable of blocking transcription when incorporated into a Ube3a-ATS minigene reporter, whereas two vector derived elements—the polyA and reverse promoter—reduced downstream transcription by up to 50%. Since not all editing events disrupt gene transcription, our findings suggest that the probability that a gene trapping AAV integration event occurs is influenced by which vector-derived element(s) are integrated and by the number of target sites.
2024-10-16 | GSE271068 | GEO
Project description:CRISPR Cas9 guided knockout (KO) of PHF6 in human THP1 AML cell line. We performed bulk RNA-Seq on knockout (PHF6 KO) and wildtype (CTRL) clones derived from THP1 cells transduced with lentiviral vectors encoding Cas9 protein and either PHF6 gRNAs or non-targeting gRNAs. Our results reveal that PHF6 knockout upregulates self-renewal gene sets and downregulates myeloid differentiation gene sets.
2024-09-18 | GSE277149 | GEO
Project description:Recombinant AAV vectors have the unique ability to promote targeted integration of transgenes via homologous integration at specified genomic sites reaching frequencies of 0.1-1%. We studied genomic parameters that influence targeting efficiencies on a large scale. To do this, we generated more than 1000 engineered, doxycycline-inducible target sites in the human HAP1 cell line and infected this polyclonal population with a library of AAV-DJ targeting vectors each carrying a unique barcode. The heterogeneity of barcode integration at each target site provided an assessment of the targeting efficiency at that locus. We compared targeting efficiency with and without target site transcription for identical chromosomal positions, finding that targeting efficiency was enhanced by target site transcription. Chromatin states associated with active transcription were also predictive of higher targeting efficiency. Furthermore, there was an effect on the amenability of a site to targeting due to other factors such as the level of transcription from intersecting genes. These results define important parameters that may not only assist in designing optimal targeting vectors for genome editing, but also provide new insights into the mechanism of AAV-mediated homologous recombination.
2020-06-04 | GSE151740 | GEO
Project description:To explore how Mier1 affects liver regeneration, we specifically knocked out Mier1 in mouse liver through adeno-associated virus (AAV). The mice we used were knocked in a Cre-induced Cas9 expression cassette. Through tail vein injection, we delivered the AAV expressing Cre-recombinase under TBG promoter, and sgRNA targeting Mier1 (AAV-Mier1 sgRNA), into the adult Cas9 knockin mice to knock out the Mier1 gene in liver. AAV vectors with no sgRNA inserted (AAV-Cre) were used in control animals. To assess the role of MIER1 in liver regeneration, we performed 70% partial hepatectomy, 3 weeks after AAV injection. We then performed ATAC-seq in Control and Mier1 liver-sepcific knockout mice liver tissues at 0 h and 24 h after PHx to explore the influence of Mier1 on the chromation opening during liver regeneration.
2022-12-31 | GSE212626 | GEO
Project description:Adenosine-to-inosine RNA base editing is a strategy developed to safely manipulate genetic information at the RNA level. Particularly promising for clinical implementation is the use of the ubiquitously expressed endogenous editing enzyme ADAR (adenosine deaminase acting on RNA) with tailored guide RNAs. However, the precision of editing could be compromised by global off-target events that can potentially occur throughout the transcriptome. In this study, we introduce a novel circular CLUSTER guide RNA design that recruits endogenous ADAR in vivo. The goals of the whole transcriptome sequencing experiment were to evaluate the A-to-G RNA editing index and the global editing precision of this novel design. To achieve this, Rett syndrome mice harboring a Mecp2 W104Amber mutation were treated either with a circular CLUSTER guide RNA targeting the mutant Mecp2 transcript or a scrambled and thus non-targeting control guide RNA. Both the targeting and the non-targeting guide RNA were encoded as AAV and delivered via retro-orbital injection of 4x10^12 viral genomes per mouse. The used AAV serotype PHP.eB allows cargo delivery to the mouse brain after systemic administration. Four weeks after injection the thalamus was isolated for NGS analysis. Whole transcriptome sequencing showed that the A-to-G RNA editing index was unaffected by treatment with the targeting guide RNA compared to the scrambled non-targeting control. We were unable to identify any global off-target events, excluding mouse to mouse variability, which suggests a very high precision of our approach on the transcriptome-wide level. Harnessing endogenous ADAR with permanent, AAV-driven CLUSTER guide RNAs in the CNS is an important next step towards the development of novel drug modalities that fight neurological diseases.
2024-06-27 | GSE265898 | GEO
Project description:CRISPR-Cas9 delivery by AAV holds promise for gene therapy but faces critical barriers due to its potential immunogenicity and limited payload capacity. Here, we demonstrate genome engineering in postnatal mice using AAV-split-Cas9, a multi-functional platform customizable for genome-editing, transcriptional regulation, and other previously impracticable AAV-CRISPR-Cas9 applications. We identify crucial parameters that impact efficacy and clinical translation of our platform, including viral biodistribution, editing efficiencies in various organs, antigenicity, immunological reactions, and physiological outcomes. These results reveal that AAV-CRISPR-Cas9 evokes host responses with distinct cellular and molecular signatures, but unlike alternative delivery methods, does not induce detectable cellular damage in vivo. Our study provides a foundation for developing effective genome therapeutics mRNA-Seq from muscles (9 samples; 3 mice x 3 conditions) and lymph nodes (9 samples; 3 mice x 3 conditions).
2016-07-29 | E-GEOD-84534 | biostudies-arrayexpress