Ontology highlight
ABSTRACT:
PROVIDER: PRJNA701945 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR13712000.fastq.gz | Fastqsanger.gz | |||
| SRR13712100.fastq.gz | Fastqsanger.gz | |||
| SRR13712200.fastq.gz | Fastqsanger.gz | |||
| SRR13712001.fastq.gz | Fastqsanger.gz | |||
| SRR13712101.fastq.gz | Fastqsanger.gz |
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Project description:Parallel genetics of regulatory sequences using scalable genome editing in vivo
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Project description:A Scalable Epitope Tagging Approach for High Throughput ChIP-seq Analysis ChIP-seq comparison between CRISPR editing cells using epitope antibody and non-editing cells using endogeneous TF antibody
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Project description:The CRISPR-Cas9 system enables efficient sequence-specific mutagenesis for creating germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-guideRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we established in vitro-assembled, fluorescent Cas9-sgRNA RNPs in stabilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. Sequence analysis of targeted loci in individual embryos reveals highly efficient bi-allelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis reveals preliminary loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show efficient targeting of functional non-coding elements in gene-regulatory regions using saturating mutagenesis towards uncovering functional control elements in transgenic reporters and endogenous genes. Our results suggest that in vitro assembled, fluorescent Cas9-sgRNA RNPs provide a rapid reverse-genetics tool for direct and scalable loss-of-function studies beyond zebrafish applications.
2016-05-01 | E-MTAB-4143 | biostudies-arrayexpress
Project description:An ability to map the global interactions of a chemical entity with chromatin genome-wide could provide new insights into the mechanisms by which a small molecule perturbs cellular functions. we developed a method that uses chemical derivatives and massively parallel DNA sequencing (Chem-Seq) to identify the sites bound by small chemical molecules throughout the human genome. We developed in vivo and in vitro Chem-Seq protocols with a biotinylated derivative of small molecules. In the in vivo protocol, Cells were first treated with biotinylated ligand and cross-linked with formaldehyde at the same time. Cells were then lysed, sonicated to shear the DNA, and streptavidin beads were used to isolate biotinylated ligand and associated chromatin fragments. We then used massively parallel sequencing to identify the enriched DNA fragments, and mapped these sequences to the genome. In in vitrol protocol, MM1.S cells were fixed and the derived sonicated lysate incubated with biotinylated drug to enrich for bound chromatin regions in vitro. We then used massively parallel sequencing to identify the enriched DNA fragments, and mapped these sequences to the genome.
2013-05-31 | E-GEOD-44098 | biostudies-arrayexpress
Project description:Advances in gene editing now allow reverse genetics to be applied to a broad range of biological systems. Ultimately, any modification to coding sequences requires confirmation at the protein level, although immunoblotting is often hampered by antibody quality or availability especially in non-mammalian species. Here, we show that Sequential Window Acquisition of All Theoretical Spectra (SWATH) - Mass Spectrometry (MS) is a robust antibody-independent alternative for monitoring gene editing at the protein level and concomitantly defines proteome responses which may provide pertinent biological insights.
2019-10-24 | PXD013176 | Pride
Project description:Prime editing enables the precise modification of genomes through reverse transcription of template sequences appended to the 3′ ends of CRISPR–Cas guide RNAs. To identify cellular determinants of prime editing, we developed scalable prime editing reporters and performed genome-scale CRISPR-interference screens. From these screens, a single factor emerged as the strongest mediator of prime editing: the small RNA-binding exonuclease protection factor La. Further investigation revealed that La promotes prime editing across approaches (PE2, PE3, PE4 and PE5), edit types (substitutions, insertions and deletions), endogenous loci and cell types but has no consistent effect on genome-editing approaches that rely on standard, unextended guide RNAs. Previous work has shown that La binds polyuridine tracts at the 3′ ends of RNA polymerase III transcripts. We found that La functionally interacts with the 3′ ends of polyuridylated prime editing guide RNAs (pegRNAs). Guided by these results, we developed a prime editor protein (PE7) fused to the RNA-binding, N-terminal domain of La. This editor improved prime editing with expressed pegRNAs and engineered pegRNAs (epegRNAs), as well as with synthetic pegRNAs optimized for La binding. Together, our results provide key insights into how prime editing components interact with the cellular environment and suggest general strategies for stabilizing exogenous small RNAs therein.
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Project description:Accurate prediction of genomic variant effects and gene expression is essential for identifying functional variations and enabling precise genome editing of cis-regulatory elements (CREs). Spatiotemporal gene expression patterns are fundamental to the formation of key traits, yet tissue-specific predictions remain inaccurate, particularly in large-genome crops like wheat. In this study, we developed DeepWheat, a suite of two models for predicting epigenomic features and gene expression in wheat. DeepEXP, a deep learning model, integrates epigenomic and transcriptomic data across various wheat tissues, achieving Pearson correlation coefficients (PCC) over 0.8 and outperforming sequence-only models, especially for tissue-specific genes. DeepEPI predicts epigenomic features from DNA sequences, helping identify regulatory sequences and facilitating model transfer across wheat varieties. Using chromatin accessibility and transcriptomic data from 9 additional wheat varieties, we validated the model’s accuracy and transfer efficiency. Our analysis further revealed that indels have a greater impact on gene expression than SNPs, and that, compared to promoter regions, the 5’UTR, 3’UTR, and introns exert even stronger regulatory effects on gene expression. These models also identified mutations that alter gene expression, supporting precise CRE editing. They provide valuable tools for tissue-specific predictions, regulatory sequence identification, and saturation mutagenesis to pinpoint high-effect sites.
2025-09-05 | GSE287695 | GEO
Project description:Accurate prediction of genomic variant effects and gene expression is essential for identifying functional variations and enabling precise genome editing of cis-regulatory elements (CREs). Spatiotemporal gene expression patterns are fundamental to the formation of key traits, yet tissue-specific predictions remain inaccurate, particularly in large-genome crops like wheat. In this study, we developed DeepWheat, a suite of two models for predicting epigenomic features and gene expression in wheat. DeepEXP, a deep learning model, integrates epigenomic and transcriptomic data across various wheat tissues, achieving Pearson correlation coefficients (PCC) over 0.8 and outperforming sequence-only models, especially for tissue-specific genes. DeepEPI predicts epigenomic features from DNA sequences, helping identify regulatory sequences and facilitating model transfer across wheat varieties. Using chromatin accessibility and transcriptomic data from 9 additional wheat varieties, we validated the model’s accuracy and transfer efficiency. Our analysis further revealed that indels have a greater impact on gene expression than SNPs, and that, compared to promoter regions, the 5’UTR, 3’UTR, and introns exert even stronger regulatory effects on gene expression. These models also identified mutations that alter gene expression, supporting precise CRE editing. They provide valuable tools for tissue-specific predictions, regulatory sequence identification, and saturation mutagenesis to pinpoint high-effect sites.
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Project description:TurboID-mediated proximity labeling of the aging and stress regulatory transcription factor DAF-16/FOXO in wild-type and daf-2(e1370) mutants in C. elegans. Endogenous DAF-16 was C-terminally tagged with the biotin ligase TurboID using CRISPR/Cas9- genome editing. In vivo proximity labeling of the DAF-16 interactome was analyzed at normal and low insulin/IGF-1 signaling conditions. The biotin-labeled DAF-16 interactome was affinity purified using streptavidin resin and analyzed with mass spec.
2025-12-29 | PXD053591 | Pride
Project description:Enhancers play important roles in evolution and disease. However, traditional assays to test enhancers are low throughput and not scalable to the >100,000 enhancers in the human genome. To better prioritize variants associated with disease and to study the role of enhancers, our group and others developed massively parallel reporter assays (MPRAs), which functionally screen thousands of sequences for regulatory activity in parallel. Although MPRAs have been applied to address diverse questions in gene regulation, there has been no systematic comparison of how differences in experimental design influence findings, making it difficult to interpret results and compare between groups. Here, we screen a library of 2,440 sequences, representing candidate liver enhancers and controls, in HepG2 cells for regulatory activity using nine different approaches (including conventional episomal, STARR-seq, and lentiviral MPRA designs). We identify subtle but significant differences in the resulting measurements that correlate with epigenetic and sequence-level features. We also test this library in both orientations with respect to the promoter, validating en masse that enhancer activity is robustly independent of orientation. Finally, we develop and apply a novel method to assemble and functionally test libraries of the same putative enhancers as 192-mers, 354-mers, and 678-mers, and observe surprisingly large differences in functional activity. This work provides a framework for the experimental design of high-throughput reporter assays, suggesting that the extended sequence context of tested elements, and to a lesser degree the precise assay, influence MPRA results.
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