Ontology highlight
ABSTRACT:
PROVIDER: PRJNA868475 | ENA |
REPOSITORIES: ENA
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Project description:CRISPR-Cas9 has tremendous potential as a therapeutic tool for treating human diseases. However, prolonged expression of the nuclease and gRNA from viral vectors in an in vivo context may cause off-target activity and immunogenicity. While extracellular vesicles have been recently demonstrated to be a promising option to transiently deliver the CRISPR-system, sufficient packaging of both Cas9 protein and gRNA is critical to achieve efficient genome editing in hard-to-transfect cells and tissues, such as skeletal muscle. Here, we developed a novel ribonucleoprotein delivery system utilizing two distinct homing mechanisms. The first is by chemical induced dimerization to recruit Cas9 protein into extracellular nanovesicles. The second utilizes a viral RNA packaging signal and two self-cleaving riboswitches to tether and release sgRNA into nanovesicles. We term our fully engineered delivery system NanoMEDIC (nanomembrane-derived extracellular vesicles for the delivery of macromolecular cargo) and demonstrate efficient genome editing in various hard-to-transfect cell types, including human iPS cells and myoblasts. Furthermore, NanoMEDIC production is scalable for industrial production as a xeno-free suspension culture system. As a disease model, therapeutic exon skipping in the dystrophin gene locus was targeted and resulted in over 90% exon skipping efficiencies in skeletal muscle cells derived from Duchenne muscular dystrophy patient iPS cells. Finally, we generated novel luciferase-based reporter mice to demonstrate that NanoMEDIC could induce exon skipping and sustain skipping activity for over 160 days, even though NanoMEDIC itself was rapidly degraded within 3 days, indicating its utility for transient in vivo genome editing therapy of DMD and beyond.
2020-01-30 | PXD014527 | JPOST Repository
Project description:Current pipelines used to map genetrap insertion sites are based on inverse- or splinkerette-PCR methods, which despite their efficacy are prone to artifacts and do not provide information on the impact of the genetrap on the expression of the targeted gene. We developed a new method, which we named TrapSeq, for the mapping of genetrap insertions based on paired-end RNA sequencing. By recognizing chimeric mRNAs containing genetrap sequences spliced to an endogenous exon, our method identifies insertions that lead to productive trapping.
2018-02-14 | GSE99483 | GEO
Project description:Chromatin structure affects gene splicing and nucleosome as the basic packaged unit is associated with exon recognition. But little is known about the function of nucleosome occupancy in the process of exon origination. Here we performed MNase-seq to obtain genome-wide nucleosome profiles for several tissues of human, rhesus monkey, tree shrew, mouse and pig. At the first time we found conserved nucleosome profile for different tissues. By combining RNA-seq data of each species, we traced the nucleosome occupancy changes of human new exons. Surprisingly, we found nucleosomes were higher occupied before exon formation. And this pre-occupancy contributed to the formation of exon-intron GC difference and maintain splice site strength. Thus we proposed a preadaption model for the function of nucleosome occupancy in human exons origination.
2018-08-07 | GSE106578 | GEO
Project description:Although CRISPR-Cas technology has revolutionized functional genomics, the systematic exploration of the role of individual exons for critical cellular phenotypes is lagging, limiting our understanding of genome regulation. To overcome this constraint, we have optimized and applied massively parallel exon deletion and splice site mutation screens in human cell lines identifying thousands of exons required for cell fitness. Fitness-promoting exons are enriched in essential and highly expressed genes and frequently overlap protein domains and interaction interfaces. This contrasts fitness-suppressing exons that are enriched in low-expressed, non-essential genes and tend to overlap intrinsically disordered regions. In-depth mechanistic investigation of a screen hit, the alternative exon-8 in TAF5, reveals that its inclusion controls the assembly of the TFIID general transcription initiation complex regulating gene expression outputs. Collectively, by applying orthogonal exon perturbation screening strategies we have interrogated phenotypically important exons at genome-scale and uncovered mechanisms that control gene expression and cell fitness.
2023-08-30 | MSV000092798 | MassIVE
Project description:PPAR-g abrogates TGF-β1 signaling by trapping the p300 co-factor in BM stromal cells
2021-04-16 | GSE141378 | GEO
Project description:Topoisomerase II (TOP2) relieves torsional stress during transcription, DNA replication and chromosome segregation, by forming transient cleavage complex intermediates (TOP2ccs) that contain TOP2-linked DNA breaks. While TOP2ccs are normally reversible they can be ‘trapped’ by chemotherapeutic drugs such as etoposide, and subsequently converted into irreversible TOP2-linked DSBs that threaten genome stability. Here, using genomics approaches, we have quantified the etoposide-induced trapping of TOP2ccs, their conversion into irreversible TOP2-linked DSBs, and their processing during DNA repair genome-wide, as a function of time. We find that while TOP2 trapping is independent of transcription it requires pre-existing binding of cohesin to DNA. In contrast, the conversion of trapped TOP2ccs to irreversible DSBs during DNA repair is accelerated two-fold at transcribed loci, relative to non-transcribed loci. This conversion is dependent on proteasomal degradation and TDP2 phosphodiesterase activity. Quantitative modeling shows that only two critical features of pre-existing chromatin structure- namely, cohesin binding and transcriptional activity- can be used to accurately predict the kinetics of TOP2-induced DSBs. Thus, our study permits a mechanistic understanding of TOP2 induced genome instability.
2019-04-26 | GSE129526 | GEO