The DNA cleavage efficiency of the hpCasMINI system [Deep-seq]
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE270587 | GEO | 2024/06/29
REPOSITORIES: GEO
Similar Datasets
Project description:The DNA cleavage efficiency of the hpCasMINI system [Deep-seq]
| PRJNA1127531 | ENA
Project description:Metal-catalyzed oxidation reactions (MCO) is one of the most applicable methods to induce protein oxidative modifications (e.g., protein oxidation, protein oxidative cleavage and crosslinking), which are essential techniques for many applications in biotechnology and redox proteomics. However, a suitable ligand is needed to assure high stability and enhance the metal catalytic efficiency in order to induce these oxidative modifications in a selective manner. Herein, we report the influence of the polyoxometalates (POMs) as inorganic ligands on the catalytic efficiency of Cu ions to induce oxidative cleavage of a protein. In the presence of ascorbic acid as (Asc), the Cu-substituted POM (Cu-POM), α2-K8P2W17O61(Cu2+.OH2), (CuWD), cleaved hen egg white lysozyme (HEWL), a protein consisting of 129 amino acids, producing only four peptide fragments. Analyzing the intact protein, in absence and presence of CuWD/Asc, and the peptide four peptide fragments via nLC-MS/MS revealed that CuWD/Asc induced oxidative modifications and cleavage at and/or near CuWD/HEWL binding sites
2023-01-30 | PXD037983 | Pride
Project description:Chemically synthesized guide RNAs can direct CRISPR-CasRx cleavage of circRNAs with high efficiency and specificity
| PRJNA785254 | ENA
Project description:Streptococcus pyogenes or group A Streptococcus (GAS) is a leading cause of bacterial pharyngitis, skin and soft tissue infections, life-threatening invasive infections, and the post-infectious autoimmune syndromes of acute rheumatic fever and post-streptococcal glomerulonephritis. Genetic manipulation of this important pathogen is complicated by resistance of the organism to genetic transformation. Very low transformation efficiency is attributed to recognition and degradation of introduced foreign DNA by a type I restriction-modification system encoded by the hsdRSM locus. DNA sequence analysis of this locus in ten GAS strains that had been previously transformed with an unrelated plasmid revealed that six of the ten harbored a spontaneous mutation in hsdR, S, or M. The mutations were all different, and at least five of the six were predicted to result in loss of function of the respective hsd gene product. The unexpected occurrence of such mutations in previously transformed isolates suggested that the process of transformation selects for spontaneous inactivating mutations in the Hsd system. We investigated the possibility of exploiting the increased transformability of hsd mutants by constructing a deletion mutation in hsdM in GAS strain 854, a clinical isolate representative of the globally dominant M1T1 clonal group. Mutant strain 854hsdM exhibited a 5-fold increase in transformation efficiency compared to the wild type parent strain and no obvious change in growth or off-target gene expression. We conclude that genetic transformation of GAS selects for spontaneous mutants in the hsdRSM restriction modification system. We propose that use of a defined hsdM mutant as a parent strain for genetic manipulation of GAS will enhance transformation efficiency and reduce the likelihood of selecting spontaneous hsd mutants with uncharacterized genotypes.
2021-04-14 | GSE167217 | GEO
Project description:Combining RNA-seq with ATAC-seq, we screened the open chromatin of dairy goat mammary epithelial cells during lactation and then integrated human lysozyme into various open regions using the CRISPR/Cas9 system. A comparison of lysozyme gene expression and integration efficiency showed that chromatin accessibility at target sites was significantly positively correlated with gene insertion efficiency, and the insertion of foreign genes into highly accessible chromatin regions could enhance its expression.
2023-12-01 | GSE225654 | GEO
Project description:We perform polyA independent deep sequencing of chromatin associated primary transcripts across three different cell lines to obtain a global view on in vivo microRNA processing. We use these data to define a MicroProcessing Index (MPI), to quantify the cleavage efficiency of the Microprocessor complex. Hallmarks of efficient Drosha-mediated processing are confirmed by means of deep sequencing of chromatin-associated transcripts upon Drosha knockdown. Our results suggest that both sequence features and thermodynamic properties, e.g. secondary structure of the regions flanking the pre-miRNA hairpins are determinants for efficient processing. Our data furthermore enables us to observe endogenous microprocessor cleavage sites at nucleotide resolution. This analysis reveals the presence of non-canonical processing events occurring one helical turn distal of most efficiently cleaved miRNA precursors. We performed polyA independent deep sequencing of the chromatin-isolated RNA fraction for 5 samples: 2 replicates in HeLa cells, 1 Drosha knock down in HeLa cells, 1 sample for A549 cells and 1 sample for HEK293 cells. We also performed deep sequencing of the small RNA fraction in the same cell lines.
2014-04-17 | E-GEOD-56862 | biostudies-arrayexpress
Project description:The CRISPR-Cas9 system has recently emerged as a versatile tool for biological and medical research. In this system, a single guide RNA (sgRNA) directs the endonuclease Cas9 to a targeted DNA sequence for site-specific manipulation. In addition to this targeting function, the sgRNA has also been shown to play a role in activating the endonuclease activity of Cas9. This dual function of the sgRNA likely underlies observations that different sgRNAs have varying on-target activities. Currently, our understanding of the relationship between sequence features of sgRNAs and their on-target cleavage efficiencies remains limited, largely due to difficulties in assessing the cleavage capacity of a large number of sgRNAs. In this study, we evaluated the cleavage activities of 218 sgRNAs using in vitro Surveyor assays. We found that nucleotides at both PAM-distal and PAM-proximal regions of the sgRNA are significantly correlated with on-target efficiency. Furthermore, we also demonstrated that the genomic context of the targeted DNA, the GC percentage, and the secondary structure of sgRNA are critical factors contributing to cleavage efficiency. In summary, our study reveals important parameters for the design of sgRNAs with high on-target efficiencies, especially in the context of high throughput applications.
| S-EPMC4728555 | biostudies-literature
Project description:In mammals, small RNAs are important players in post-transcriptional gene regulation. While their roles in mRNA destabilization and translational repression are well appreciated, their involvement in endonucleolytic cleavage of target RNAs is poorly understood. Very few microRNAs are known to guide RNA cleavage. Endogenous small interfering RNAs are expected to induce target cleavage, but their target genes remain largely unknown. We report a systematic study of small RNA-mediated endonucleolytic cleavage in mouse through integrative analysis of small RNA and degradome sequencing data without imposing any bias towards known small RNAs. Hundreds of small cleavage-inducing RNAs and their cognate target genes were identified, significantly expanding the repertoire of known small RNA-guided cleavage events. Strikingly, both small RNAs and their target sites demonstrated significant overlap with retrotransposons, providing evidence for the long-standing speculation that retrotransposable elements in mRNAs are leveraged as signals for gene targeting. Furthermore, our analysis showed that the RNA cleavage pathway is also present in human cells but affecting a different repertoire of retrotransposons. These results show that small RNA-guided cleavage is more widespread than previously appreciated. Their impact on retrotransposons in non-coding regions shed light on important aspects of mammalian gene regulation. rRNA depleted RNA sequencing was done for four stages of testis development.
2016-03-16 | E-GEOD-68251 | biostudies-arrayexpress
Project description:In mammals, small RNAs are important players in post-transcriptional gene regulation. While their roles in mRNA destabilization and translational repression are well appreciated, their involvement in endonucleolytic cleavage of target RNAs is poorly understood. Very few microRNAs are known to guide RNA cleavage. Endogenous small interfering RNAs are expected to induce target cleavage, but their target genes remain largely unknown. We report a systematic study of small RNA-mediated endonucleolytic cleavage in mouse through integrative analysis of small RNA and degradome sequencing data without imposing any bias towards known small RNAs. Hundreds of small cleavage-inducing RNAs and their cognate target genes were identified, significantly expanding the repertoire of known small RNA-guided cleavage events. Strikingly, both small RNAs and their target sites demonstrated significant overlap with retrotransposons, providing evidence for the long-standing speculation that retrotransposable elements in mRNAs are leveraged as signals for gene targeting. Furthermore, our analysis showed that the RNA cleavage pathway is also present in human cells but affecting a different repertoire of retrotransposons. These results show that small RNA-guided cleavage is more widespread than previously appreciated. Their impact on retrotransposons in non-coding regions shed light on important aspects of mammalian gene regulation. Refer to individual Series
2016-03-16 | E-GEOD-68254 | biostudies-arrayexpress