Ontology highlight
ABSTRACT:
PROVIDER: PRJNA998575 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR25422501_1.fastq.gz | Fastqsanger.gz | |||
| SRR25422501_2.fastq.gz | Fastqsanger.gz | |||
| SRR25422502_1.fastq.gz | Fastqsanger.gz | |||
| SRR25422502_2.fastq.gz | Fastqsanger.gz | |||
| SRR25422503_1.fastq.gz | Fastqsanger.gz |
Items per page: 5 1 - 5 of 30 |
Similar Datasets
Project description:Assaying Cas9 HDR efficiency in human cell line
| PRJNA436691 | ENA
Project description:This SuperSeries is composed of the following subset Series: GSE29800: Lack of G1 cyclin arrest cell cycle synchronization time-course microarray in Glucose GSE29892: Alpha Factor arrest cell cycle synchronization time-course microarray in Galactose GSE29893: Alpha Factor arrest cell cycle synchronization time-course microarray in Glucose Refer to individual Series
2011-06-11 | E-GEOD-29894 | biostudies-arrayexpress
Project description:By applying a barcoding strategy to clonal tracking of edited cells (BAR-seq), we show that p53 activation triggered by HDR editing significantly shrink the HSPC clonal repertoire in hematochimeric mice, despite engrafted edited clones preserved multilineage and self-renewing capacity. Transient inhibition of p53 restored polyclonal graft composition. We then overcame HSC constraints to HDR by forcing cell cycle progression and upregulating components of the HDR machinery through transient expression of Adenovirus 5 E4orf6/7 protein, which operates the major cell cycle controller E2F. Combined E4orf6/7 expression and p53 inhibition resulted in high and stable HDR editing efficiencies in the human graft, without perturbing repopulation and self-renewing properties of edited HSCs.
2020-04-22 | GSE143995 | GEO
Project description:By applying a barcoding strategy to clonal tracking of edited cells (BAR-seq), we show that p53 activation triggered by HDR editing significantly shrink the HSPC clonal repertoire in hematochimeric mice, despite engrafted edited clones preserved multilineage and self-renewing capacity. Transient inhibition of p53 restored polyclonal graft composition. We then overcame HSC constraints to HDR by forcing cell cycle progression and upregulating components of the HDR machinery through transient expression of Adenovirus 5 E4orf6/7 protein, which operates the major cell cycle controller E2F. Combined E4orf6/7 expression and p53 inhibition resulted in high and stable HDR editing efficiencies in the human graft, without perturbing repopulation and self-renewing properties of edited HSCs.
2020-04-22 | GSE144340 | GEO
Project description:Enhancing site-specific transgene insertion efficiency by homology-directed repair (HDR) using high concentrations of double-stranded DNA (dsDNA) with Cas9 target sequences (CTSs) can be toxic to primary cells. Here, we develop single-stranded DNA (ssDNA) HDR templates (HDRTs) incorporating CTSs with reduced toxicity that boost knock-in efficiency and yield by an average of 2–3-fold relative to dsDNA CTSs. Using small-molecule combinations that enhance HDR, we could further increase knock-in efficiencies by an additional 2–3-fold on average. Our method works across a variety of target loci, knock-in constructs, and primary human cell types, reaching HDR efficiencies of >80–90%. We demonstrate application of this approach for both pathogenic gene variant modeling and gene-replacement strategies for IL2RA and CTLA4 mutations associated with Mendelian disorders. Finally, we develop a good manufacturing practice (GMP)-compatible process for non-viral chimeric antigen receptor (CAR)-T cell manufacturing, with knock-in efficiencies (46–62%) and yields (>1.5×109 modified cells) exceeding those of conventional approaches.
2022-05-21 | GSE203349 | GEO
Project description:Enhancing site-specific transgene insertion efficiency by homology-directed repair (HDR) using high concentrations of double-stranded DNA (dsDNA) with Cas9 target sequences (CTSs) can be toxic to primary cells. Here, we develop single-stranded DNA (ssDNA) HDR templates (HDRTs) incorporating CTSs with reduced toxicity that boost knock-in efficiency and yield by an average of 2–3-fold relative to dsDNA CTSs. Using small-molecule combinations that enhance HDR, we could further increase knock-in efficiencies by an additional 2–3-fold on average. Our method works across a variety of target loci, knock-in constructs, and primary human cell types, reaching HDR efficiencies of >80–90%. We demonstrate application of this approach for both pathogenic gene variant modeling and gene-replacement strategies for IL2RA and CTLA4 mutations associated with Mendelian disorders. Finally, we develop a good manufacturing practice (GMP)-compatible process for non-viral chimeric antigen receptor (CAR)-T cell manufacturing, with knock-in efficiencies (46–62%) and yields (>1.5×109 modified cells) exceeding those of conventional approaches.
2022-05-12 | GSE202596 | GEO
Project description:Enhancing site-specific transgene insertion efficiency by homology-directed repair (HDR) using high concentrations of double-stranded DNA (dsDNA) with Cas9 target sequences (CTSs) can be toxic to primary cells. Here, we develop single-stranded DNA (ssDNA) HDR templates (HDRTs) incorporating CTSs with reduced toxicity that boost knock-in efficiency and yield by an average of 2–3-fold relative to dsDNA CTSs. Using small-molecule combinations that enhance HDR, we could further increase knock-in efficiencies by an additional 2–3-fold on average. Our method works across a variety of target loci, knock-in constructs, and primary human cell types, reaching HDR efficiencies of >80–90%. We demonstrate application of this approach for both pathogenic gene variant modeling and gene-replacement strategies for IL2RA and CTLA4 mutations associated with Mendelian disorders. Finally, we develop a good manufacturing practice (GMP)-compatible process for non-viral chimeric antigen receptor (CAR)-T cell manufacturing, with knock-in efficiencies (46–62%) and yields (>1.5×109 modified cells) exceeding those of conventional approaches.
2022-05-21 | GSE202909 | GEO
Project description:The changes in global gene expression in response to DNA damage may derive from either direct induction or repression by transcriptional regulation or indirectly by synchronization of cells to specific cell cycle phases, such as G1 or G2. We developed a model that successfully estimated the expression levels of >400 cell-cycle-regulated genes in normal human fibroblasts based on the proportions of cells in each phase of the cell cycle. By isolating effects on the gene expression associated with the cell cycle phase redistribution after genotoxin treatment, the direct transcriptional target genes were distinguished from genes for which expression changed secondary to cell synchronization. Application of this model to ionizing radiation (IR)-treated normal human fibroblasts identified 150 of 406 cycle-regulated genes as putative direct transcriptional targets of IR-induced DNA damage. Changes in expression of these genes after IR treatment derived from both direct transcriptional regulation and cell cycle synchronization. Keywords: Microarray, Cell cycle, Ionizing radiation, Human fibroblasts, EPIG
2007-08-01 | GSE7075 | GEO
Project description:Homology Directed Repair (HDR) enables precise genome editing and holds great promise in the gene therapy field. However, the implementation of HDR-based therapies is hindered by limited efficiency in comparison to methods that exploit alternative DNA repair routes, such as Non-Homologous End Joining (NHEJ). In this study, we demonstrate the development of a functional, pooled screening platform utilizing an HDR-based readout to identify protein-based reagents that improve HDR outcomes in human hematopoietic stem and progenitor cells (HSPCs), a clinically relevant cell type for gene therapy. We leveraged this screening platform to explore sequence diversity at the binding interface of the NHEJ inhibitor i53 and its target, 53BP1, and we identified optimized i53 variants that enable new intermolecular bonds and robustly increase HDR. These variants specifically reduce insertion-deletion outcomes and also synergize with a DNAPK inhibitor to increase HDR rates. When applied at manufacturing scale, the incorporation of improved variants results in a significant increase in cells with at least one repaired allele and improved HDR in long-term HSPCs subpopulations, while not increasing off-target editing or gross chromosomal rearrangements. We anticipate the pooled screening platform will enable discovery of future gene editing reagents that improve HDR outcomes, such as the i53 variants reported here.
2024-02-11 | GSE242749 | GEO