Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1003052 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR25549500.fastq.gz | Fastqsanger.gz | |||
| SRR25549600.fastq.gz | Fastqsanger.gz | |||
| SRR25549700.fastq.gz | Fastqsanger.gz | |||
| SRR25549501.fastq.gz | Fastqsanger.gz | |||
| SRR25549601.fastq.gz | Fastqsanger.gz |
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Project description:Genome editing was conducted on a t(3;8) K562 model to investigate the effects of deleting different modules or CTCF binding sites within the MYC super-enhancer. To check mutations after targeting with CRISPR-Cas9 we performed amplicon sequencing using the Illumina PCR-based custom amplicon sequencing method using the TruSeq Custom Amplicon index kit (Illumina). The first PCR was performed using Q5 polymerase (NEB), the second nested PCR with KAPA HiFi HotStart Ready mix (Roche). Samples were sequenced paired-end (2x 250bp) on a MiSeq (Illumina).
2021-07-26 | E-MTAB-9937 | biostudies-arrayexpress
Project description:Purpose: Chinese hamster ovary (CHO) cells are a common tool utilized in bioproduction and directed genome engineering of CHO cells is of great interest to enhance recombinant cell lines. Until recently, this focus has been challenged by a lack of efficacious, high throughput, and low-cost gene editing modalities and screening methods. In this work, we demonstrate an improved method for gene editing in CHO cells using CRISPR RNPs and characterize the endpoints of Cas9 and ZFN mediated genetic engineering. Methods: Amplicons were first assayed for quality using an Invitrogen Quant-iT dsDNA (Thermo, Cat Q33120) assay and gel electrophoresis to determine DNA concentration and DNA quality. Samples were then used for Sanger sequence based decomposition or processed downstream using NGS. For NGS, libraries were generated using the Illumina TruSeq Nano DNA kit (Illumina, Cat: 20015964). The libraries were sequenced using the Illumina MiSeq platform, with read length of 2x150bp. 4.5Gb of sequencing data was generated per DNA sample. Results: There was a tendency for decomposition to underestimate editing as compared to NGS as the indel size became larger. Despite these results, sequence decomposition was able to reproduce next-generation data in the context of gene editing efficiency across a short amplicon. Conclusions: We took advantage of the differential Indel spectrums induced by both ZFN and CRISPR to validate metrics for assess gene editing and show how deconvolution-based methods, can be utilized in the context of cell line development.
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Project description:To compare the impact of CRISPR-egineered R175 TP53 mutant variants in HCT116 and H460 cells, mutations at the amino acid position 175 were generated systematically by CRISP/Cas9 editing. Here, genomic amplicon regions covering the TP53 Exons 5 were sequenced via targeted sequencing.
2024-10-16 | E-MTAB-14322 | biostudies-arrayexpress
Project description:CRISPR/Cas9-based genome editing has revolutionized experimental molecular biology and entered the clinical world for targeted gene therapy. Identifying DNA modifications occurring at CRISPR/Cas9 target sites is critical to determine efficiency and safety of editing tools. Here we show that insertions of LINE-1 (L1) retrotransposons can occur frequently at CRISPR/Cas9 editing sites. Together with PolyA-seq and an improved amplicon sequencing, we characterize more than 2,500 de novo L1 insertions at multiple CRISPR/Cas9 editing sites in HEK293T, HeLa and U2OS cells. These L1 retrotransposition events exploit CRISPR/Cas9-induced DSB formation and require L1 RT activity. Importantly, de novo L1 insertions are rare during genome editing by prime editors (PE), cytidine or adenine base editors (CBE or ABE), consistent with their reduced DSB formation. These data demonstrate that insertions of retrotransposons might be a potential outcome of CRISPR/Cas9 genome editing and provide further evidence on the safety of different CRISPR-based editing tools.
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Project description:Aims: A mutation in the phospholamban (PLN) gene, leading to deletion of Arg14 (R14del), has been associated with malignant arrhythmias and ventricular dilation. Identifying pre-symptomatic carriers with vulnerable myocardium is crucial because arrhythmia can result in sudden cardiac death, especially in young adults with PLN-R14del mutation. This study aimed at assessing the efficiency and efficacy of in vivo genome editing, using CRISPR/Cas9 and a cardiotropic adeno-associated virus (AAV9), in improving cardiac function in young adult mice expressing the human PLN-R14del. Methods and Results: Humanized mice were generated expressing human wild-type (hPLN-WT) or mutant (hPLN-R14del) PLN in the heterozygous state, mimicking human carriers. Cardiac magnetic resonance imaging at 12 weeks of age showed bi-ventricular dilation and increased stroke volume in mutant vs. WT mice, with no deficit in ejection fraction or cardiac output. Challenge of ex vivo hearts with isoproterenol and rapid pacing unmasked higher propensity for sustained ventricular tachycardia (VT) in hPLN-R14del relative to hPLN-WT. Specifically, the VT threshold was significantly reduced (20.3±1.2 Hz in hPLN-R14del vs. 25.7±1.3 Hz in WT, p<0.01) reflecting higher arrhythmia burden. To inactivate the R14del allele, mice were tail-vein-injected with AAV9.CRISPR/Cas9/gRNA or AAV9 empty capsid (controls). CRISPR-Cas9 efficiency was evaluated by droplet digital PCR and NGS-based amplicon sequencing. In vivo gene editing significantly reduced end diastolic and stroke volumes in hPLN-R14del CRISPR-treated mice compared to controls. Susceptibility to VT was also reduced, as the VT threshold was significantly increased relative to controls (30.9±2.3 Hz vs. 21.3±1.5 Hz; p<0.01). Conclusions: This study is the first to show that disruption of hPLN-R14del allele by AAV9- CRISPR/Cas9 improves cardiac function and reduces VT susceptibility in humanized PLN-R14del mice, offering preclinical evidence for translatable approaches to therapeutically suppress the arrhythmogenic phenotype in human patients with PLN-R14del disease.
2023-03-11 | PXD031612 | Pride
Project description:Many previous studies, including the Next Generation Sequencing (NGS)-based ones, have shown the critical roles of RNA editing in biomedicine. Direct RNA sequencing emerges as another powerful technique to advance the understanding of RNA editing by new paradigms, especially in single-molecule and long-range characterization. The urgent gap is the accurate and robust identification of RNA editing at the single-molecule and single-nucleotide resolution from direct RNA sequencing. This is challenging due to the inherent nature of the context-dependence on the raw signals, which requires enormous training data with considerable diversity. Here we propose two coupled measures to address them: 1) an abductive deep learning strategy implemented as the software ReDD fully utilizes the widely accessible NGS-based RNA editing data as indirect labels of direct RNA sequencing to achieve the detection at the single-molecule level; 2) a cloud-based platform Argo-ReDD serves as a central database for assembling large and diverse data from the community to continuously train the abductive deep learning model, which also meets the community demand of a user-friendly way to perform RNA editing analyses, such as co-occurrence analysis, quantitative analysis and gene isoform-resolved analysis, based on the specific information from direct RNA sequencing.
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