Homo sapiens
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA276663 | ENA |
REPOSITORIES: ENA
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Project description:Genome editing with programmable nucleases has shown great promise for clinical translation but also revealed the risk of genotoxicity caused by chromosomal translocations or the insertion of mutations at off-target sites. Here, we describe CAST-Seq, an innovative assay to identify and quantify chromosomal aberrations derived from on- and off-target activities of CRISPR-Cas nucleases or TALENs. CAST-Seq also detected novel types of chromosomal rearrangements, including homology-mediated translocations that are mediated by homologous recombination. Depending on the employed designer nuclease, translocations occurred in 0–0.5% of gene-edited human stem cells and some 20% of target loci harbored gross aberrations. In conclusion, CAST-Seq analyses are particularly relevant for therapeutic editing of stem cells to enable a thorough risk assessment before clinical application of gene editing products.
2021-02-14 | GSE164389 | GEO
Project description:Programmable nucleases and designer-recombinases are prominent genome editing tools that hold great potential for the treatment of human genetic disorders. However, both of these tools alone are not optimal for clinical applications. We present an approach that combines the ease of targeting of programmable nucleases with editing safety and accuracy of site-specific recombinases. We find that insertional fusions of zinc-finger DNA-binding domains (ZFDs) into the coding sequence of designer-recombinases generate conditional enzymes that are inactive, unless the ZFD binds its target site placed in the vicinity of the recombinase binding site. This induced-fit activity is transferable to a recombinase with relaxed specificity, representing the prototype of a new class of genome editing enzymes that opens exciting perspectives for flexible, seamless, and precise genome surgery.
2023-12-19 | GSE243676 | GEO
Project description:X-CGD (X-linked chronic granulomatous disease) patient derived CD34+ cells were reprogrammed into two X-CGD iPSC clones (7 and 25) and subsequently corrected using ZFN (clone 7) and TALEN (clone 25) by integrating a therapeutic gp91phox expression cassette into the AAVS1 "safe harbor" locus Three condition experiment: before and after reprogramming and before and after genome editing.
2015-02-28 | E-GEOD-66337 | biostudies-arrayexpress
Project description:Genome editing research of human ES/iPS cells has been accelerated by clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) and transcription activator-like effector nucleases (TALEN) technologies. However, the efficiency of biallelic genetic engineering in transcriptionally inactive genes is still low, unlike that in transcriptionally active genes. To enhance the biallelic homologous recombination efficiency in human ES/iPS cells, we performed screenings of accessorial genes and compounds. We found that RAD51 overexpression and valproic acid treatment enhanced biallelic-targeting efficiency in human ES/iPS cells regardless of the transcriptional activity of the targeted locus. Importantly, RAD51 overexpression and valproic acid treatment synergistically increased the biallelic homologous recombination efficiency. Our findings would facilitate genome editing study using human ES/iPS cells.
2017-03-26 | GSE69653 | GEO
Project description:We aimed to identify the effect of AGO2 depletion by genome editing on the mRNA expression profile of human cells. The experimental model used is the human cell line HeLaS3. All copies of AGO2 gene were disrupted by targeted genome editing using ZNF nucleases.
2019-01-28 | E-MTAB-7085 | biostudies-arrayexpress
Project description:Chronic granulomatous disease (CGD) is a severe inborn error of immunity (IEI) caused by defects in the NADPH oxidase. The best current treatment option for patients with CGD, allogeneic hematopoietic stem cell (HSC) transplantation, is associated with severe adverse effects such as graft-versus-host disease, highlighting a need for improved treatment options based on transplantation of autologous ex vivo gene-edited HSCs. Here, we generate CRISPR/Cas9-based gene editing strategies for the correction of two CGD-causing variants; CYBA c.287+1G>T (p22phox) causing autosomal CGD and CYBB c.252G>A (NOX2) causing X-linked CGD. We find that rAAV6 outperforms ssODNs and IDLVs as HDR repair templates in CD34+ hematopoietic stem and progenitor cells (HSPCs), and we optimize gene editing strategies further by including mRNA-encoded inhibitors/effectors. In addition, we develop a near-universal gene editing strategy for X-CGD by targeted integration of a truncated CYBB cDNA, covering 86% of X-CGD patients, and show functional ROS production in edited cells. We find prevalent off-target editing and chromosomal translocations that severely decreased the ability of gene edited HSPCs to engraft in immunodeficient mice, however, by limiting off-target editing through the use of a high-fidelity Cas9, we can show that we can substantially rescue the multilineage engraftment potential of the gene edited HSPCs. To further improve safety, we finally we develop a novel paired D10A Cas9n gene editing approach targeting CYBB. We demonstrate that using this D10A Cas9 approach, we can retain high on-target efficacy while we do not detect any off-target editing or translocations between on- or off-target cleavage sites. Collectively, we bring new insights into to safety and off-target effects of gene editing approaches targeting CYBA and CYBB, highlighting key challenges of these approaches while offering a potentially curative D10A Cas9n-based treatment option for patients with CGD with improved safety.
2025-02-24 | GSE287370 | GEO
Project description:Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.
| S-EPMC2531291 | biostudies-literature
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs.
2010-09-25 | GSE24338 | GEO
Project description:RNA extracted at 240min. Samples are rRNA depleted. Expression measurement of Homo sapiens genes.
2019-07-31 | GSE114179 | GEO
Project description:Transcriptional profiling of human mesenchymal stem cells comparing normoxic MSCs cells with hypoxic MSCs cells. Hypoxia may inhibit senescence of MSCs during expansion. Goal was to determine the effects of hypoxia on global MSCs gene expression.
2011-02-19 | GSE20463 | GEO