Gene editing of colocalized NCF1 loci is associated with homologous recombination and chromosomal rearrangements - CAST-seq
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1098731 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:Despite lingering genotoxicity concerns, CRISPR-based genome editing has progressively entered the gene therapy field. Here we report that interactions between nucleotide sequences that are localized on the same chromosome and exhibit a high degree of homology contribute substantially to post-editing chromosomal rearrangements. We successfully employed corrective editing strategies at the NCF1 gene, as well as at the colocalized pseudogenes, NCF1B and NCF1C, in a human cell line model of p47phox-deficient chronic granulomatous disease and in patient-derived human hematopoietic stem cells. Upon genetic manipulation, an improved droplet digital PCR-based method identified cells with altered copy numbers, spanning megabases from the edited loci. We attributed the high aberration frequency to the interaction between repetitive sequences and their predisposition to recombination events. Our findings emphasize the need for careful evaluation of the target-specific genomic context, such as the presence of homologous regions, whose genome instability can constitute a risk factor for chromosomal rearrangements upon genome editing.
2024-10-16 | GSE263692 | GEO
Project description:Gene editing of colocalized NCF1 loci is associated with homologous recombination and chromosomal rearrangements
| PRJNA1123171 | ENA
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:This SuperSeries is composed of the SubSeries listed below.
2024-10-18 | GSE269656 | GEO
Project description:At the organismal level, genome rearrangements are usually deleterious and are often associated with disease. Yet, on an evolutionary scale, they can be beneficial as they provide for rapid genetic diversification. DNA lesions, particularly double-strand breaks (DSBs), are sources of genome instability that can be rectified by various repair processes. Homologous recombination (HR) is highly effective at protecting the genome from DSBs and provides for accurate repair between sister chromatids and homologous chromosomes. Here we show that although random DSBs induced by ionizing radiation in yeast chromosomes are repaired efficiently by HR in G-2 diploid cells, rearrangements are frequent. The chromosome aberrations (ABs) primarily resulted from recombination between Ty retrotransposable elements, the most abundant class of dispersed repetitive DNAs in the genome, while some rearrangements involved other classes of repetitive DNA. Few, if any, of the ABs could be attributed to nonhomologous end-joining (NHEJ). We conclude that only those few DSBs that fall at or near the 3-5% of the genome composed of repetitive DNA elements are effective at generating rearrangements, while other lesions that appear in unique (single copy) sequences are correctly repaired. Thus, by successfully competing with repair that normally occurs between large homologous chromosomal DNAs, the combination of repetitive elements and DSBs provides genome plasticity and a rich source of evolutionary opportunities. Keywords: CGH-array
2007-02-08 | GSE6984 | GEO
Project description:Quantitative evaluation of chromosomal rearrangements in primary gene-edited human stem cells by preclinical CAST-Seq
| PRJNA690516 | ENA
Project description:At the organismal level, genome rearrangements are usually deleterious and are often associated with disease. Yet, on an evolutionary scale, they can be beneficial as they provide for rapid genetic diversification. DNA lesions, particularly double-strand breaks (DSBs), are sources of genome instability that can be rectified by various repair processes. Homologous recombination (HR) is highly effective at protecting the genome from DSBs and provides for accurate repair between sister chromatids and homologous chromosomes. Here we show that although random DSBs induced by ionizing radiation in yeast chromosomes are repaired efficiently by HR in G-2 diploid cells, rearrangements are frequent. The chromosome aberrations (ABs) primarily resulted from recombination between Ty retrotransposable elements, the most abundant class of dispersed repetitive DNAs in the genome, while some rearrangements involved other classes of repetitive DNA. Few, if any, of the ABs could be attributed to nonhomologous end-joining (NHEJ). We conclude that only those few DSBs that fall at or near the 3-5% of the genome composed of repetitive DNA elements are effective at generating rearrangements, while other lesions that appear in unique (single copy) sequences are correctly repaired. Thus, by successfully competing with repair that normally occurs between large homologous chromosomal DNAs, the combination of repetitive elements and DSBs provides genome plasticity and a rich source of evolutionary opportunities. Keywords: Band-array
2007-02-10 | GSE6991 | GEO
Project description:At the organismal level, genome rearrangements are usually deleterious and are often associated with disease. Yet, on an evolutionary scale, they can be beneficial as they provide for rapid genetic diversification. DNA lesions, particularly double-strand breaks (DSBs), are sources of genome instability that can be rectified by various repair processes. Homologous recombination (HR) is highly effective at protecting the genome from DSBs and provides for accurate repair between sister chromatids and homologous chromosomes. Here we show that although random DSBs induced by ionizing radiation in yeast chromosomes are repaired efficiently by HR in G-2 diploid cells, rearrangements are frequent. The chromosome aberrations (ABs) primarily resulted from recombination between Ty retrotransposable elements, the most abundant class of dispersed repetitive DNAs in the genome, while some rearrangements involved other classes of repetitive DNA. Few, if any, of the ABs could be attributed to nonhomologous end-joining (NHEJ). We conclude that only those few DSBs that fall at or near the 3-5% of the genome composed of repetitive DNA elements are effective at generating rearrangements, while other lesions that appear in unique (single copy) sequences are correctly repaired. Thus, by successfully competing with repair that normally occurs between large homologous chromosomal DNAs, the combination of repetitive elements and DSBs provides genome plasticity and a rich source of evolutionary opportunities. Keywords: CGH-array Diploid G-2 yeast cells were exposed to 80 krad of ionizing radiation and plated on rich media to obtain survivor colonies. Genomic DNA from each of 37 survivors (Cy5/red; JW1 to JW13, and A1 to A24) was competitively hybridized to DNA from the parent diploid strain (Cy3/green). Gains of genomic segments in the survivors were detected as continuous regions of positive Log2 Red:Green ratios, while losses were detected as negative Log2 Red:Green ratios.
2010-06-11 | E-GEOD-6984 | biostudies-arrayexpress
Project description:The Mec1/ATR kinase is crucial for genome stability, yet the mechanism by which it prevents gross chromosomal rearrangements (GCRs) remains unknown. Here we find that in cells with deficient Mec1 signaling, GCRs accumulate due to the deregulation of multiple steps in homologous recombination (HR). Mec1 primarily suppresses GCRs through its role in activating the canonical checkpoint kinase Rad53, which ensures the proper control of DNA end resection. Upon loss of Rad53 signaling and resection control, Mec1 becomes hyperactivated and triggers a salvage pathway in which the Sgs1 helicase is recruited to sites of DNA lesions via the 911-Dpb11 scaffolds and phosphorylated by Mec1 to favor heteroduplex rejection and limit HR-driven GCR accumulation. Fusing an ssDNA recognition domain to Sgs1 bypasses the requirement of Mec1 signaling for GCR suppression and nearly eliminates D-loop formation, thus preventing non-allelic recombination events. We propose that Mec1 regulates multiple steps of HR to prevent GCRs while ensuring balanced HR usage when needed for promoting tolerance to replication stress.
| S-SCDT-10_1038-S44318-024-00139-9 | biostudies-other
Project description:At the organismal level, genome rearrangements are usually deleterious and are often associated with disease. Yet, on an evolutionary scale, they can be beneficial as they provide for rapid genetic diversification. DNA lesions, particularly double-strand breaks (DSBs), are sources of genome instability that can be rectified by various repair processes. Homologous recombination (HR) is highly effective at protecting the genome from DSBs and provides for accurate repair between sister chromatids and homologous chromosomes. Here we show that although random DSBs induced by ionizing radiation in yeast chromosomes are repaired efficiently by HR in G-2 diploid cells, rearrangements are frequent. The chromosome aberrations (ABs) primarily resulted from recombination between Ty retrotransposable elements, the most abundant class of dispersed repetitive DNAs in the genome, while some rearrangements involved other classes of repetitive DNA. Few, if any, of the ABs could be attributed to nonhomologous end-joining (NHEJ). We conclude that only those few DSBs that fall at or near the 3-5% of the genome composed of repetitive DNA elements are effective at generating rearrangements, while other lesions that appear in unique (single copy) sequences are correctly repaired. Thus, by successfully competing with repair that normally occurs between large homologous chromosomal DNAs, the combination of repetitive elements and DSBs provides genome plasticity and a rich source of evolutionary opportunities. Keywords: Band-array Each array in this series corresponds to the DNA of a yeast chromosomal band excised from a pulse-field gel (CHEF). Chromosomal aberrations identified in radiation survivors were analyzed by microarray to reveal which regions of the genome were present in the new chromosomes. The DNA enriched in the specific band appears in the arrays as continuos segments of spots with highly positive Log2 Red/Green ratios.
2010-06-11 | E-GEOD-6991 | biostudies-arrayexpress