Kinetics and fidelity of the repair of Cas9-induced double-strand DNA breaks
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE113129 | GEO | 2018/05/01
REPOSITORIES: GEO
Similar Datasets
Project description:DNA double strand breaks (DSBs) are a major source of mutations. Both non-homologous-end-joining (NHEJ) and microhomology-mediated-end-joining (MMEJ) DSB repair pathways are error prone and produce deletions, which can lead to cancer. DSBs also lead to epigenetic changes, including demethylation, which is involved in carcinogenesis. Of specific interest is the MMEJ repair pathway, as it requires methylation restoration around the break, as a result of the resection and formation of single stranded (ssDNA) intermediates. While, methylation patterns after homologous recombination (HR) have been partially studied, the methylation status after MMEJ and NHEJ remains poorly reported, and can be relevant for cancer. To study methylation patterns around DSB after NHEJ and MMEJ repair, we used targeted bisulfite-sequencing (BS-seq) to quantify methylation of dozens of single cell clones after induction of DSB by CRISPR. Each single cell clone was classified according to the sequence signature to a specific repair mechanism: NHEJ or MMEJ. Comparison of single cell clones after DSB to control cells, without DSB, demonstrated correct restoration of the methylation levels. No difference in methylation patterns was noticed when comparing NHEJ to MMEJ. Methylation levels in gene body, highly methylated CpGs (n=61, 4000 base pairs around DSB) and in low methylation CpGs (n=19), remained stable after both MMEJ and NHEJ. Gene body methylation persisted even on the background of DNMT3A R882C mutation, the most prevalent preleukemic mutation, in which the de novo methylation machinery is compromised. An exception observed in a single CpG site (ASXL1 995) which demonstrated elevated methylation rate after DSB repair only in the presence of WT DNMT3A. In summary, DNA methylation restoration demonstrated high fidelity after DSB both in methylated and unmethylated gene body, even in cases where DNA resections and deletions occurred.
2022-09-11 | E-MTAB-12037 | biostudies-arrayexpress
Project description:DNA double strand breaks (DSBs) are a major source of mutations. Both non-homologous-end-joining (NHEJ) and microhomology-mediated-end-joining (MMEJ) DSB repair pathways are error prone and produce deletions, which can lead to cancer. DSBs also lead to epigenetic changes, including demethylation, which is involved in carcinogenesis. Of specific interest is the MMEJ repair pathway, as it requires methylation restoration around the break, as a result of the resection and formation of single stranded (ssDNA) intermediates. While, methylation patterns after homologous recombination (HR) have been partially studied, the methylation status after MMEJ and NHEJ remains poorly reported, and can be relevant for cancer. To study methylation patterns around DSB after NHEJ and MMEJ repair, we used targeted bisulfite-sequencing (BS-seq) to quantify methylation of dozens of single cell clones after induction of DSB by CRISPR. Each single cell clone was classified according to the sequence signature to a specific repair mechanism: NHEJ or MMEJ. Comparison of single cell clones after DSB to control cells, without DSB, demonstrated correct restoration of the methylation levels. No difference in methylation patterns was noticed when comparing NHEJ to MMEJ. Methylation levels in gene body, highly methylated CpGs (n=61, 4000 base pairs around DSB) and in low methylation CpGs (n=19), remained stable after both MMEJ and NHEJ. Gene body methylation persisted even on the background of DNMT3A R882C mutation, the most prevalent preleukemic mutation, in which the de novo methylation machinery is compromised. An exception observed in a single CpG site (ASXL1 995) which demonstrated elevated methylation rate after DSB repair only in the presence of WT DNMT3A. In summary, DNA methylation restoration demonstrated high fidelity after DSB both in methylated and unmethylated gene body, even in cases where DNA resections and deletions occurred.
2022-08-25 | E-MTAB-12061 | biostudies-arrayexpress
Project description:The structure of broken DNA ends is a critical determinant of the pathway used for DNA double strand break (DSB) repair. Here, we develop an approach, hairpin capture of DNA end structures (HCoDES), which elucidates chromosomal DNA end structures at single nucleotide resolution. HCoDES defines structures of physiologic DSBs generated by the RAG endonuclease, as well as those generated by nucleases widely used for genome editing. Analysis of G1-phase cells deficient in H2AX or 53BP1 reveals DNA ends that are frequently resected to form long single-stranded overhangs that can be repaired by mutagenic pathways. In addition to 3’ overhangs, many of these DNA ends unexpectedly form long 5’ single-stranded overhangs. The divergence in DNA end structures resolved by HCoDES suggests that H2AX and 53BP1 may have distinct activities in end protection. Thus, the high-resolution end structures obtained by HCoDES identify new features of DNA end processing during DSB repair. Single stranded DNA ligation of genomic DNA isolated from G1 arrested LigaseIV-/-, LigaseIV-/- 53BP1-/- and LigaseIV-/- H2AX-/- Abelson pre-B cells harboring site specific DSBs generated by the RAG recombinase, Cas9 endonuclease or Zinc Finger Endonuclease.
2014-12-03 | E-GEOD-62534 | biostudies-arrayexpress
Project description:Cells have developed effective mechanisms, namely homologous recombination (HR) and non-homologous end-joining (NHEJ), to repair DNA double-strand breaks (DSBs), which are considered to be the most deleterious type of damage that can challenge genome integrity. While these pathways coexist to repair DSBs, the mechanisms by which one of these pathways is chosen to repair a particular DSB remain unclear. Here, we show that the chromatin context in which a break occurs participates in this choice and that transcriptionnaly active chromatin channels repair to HR. By using a human cell line expressing a restriction enzyme fused to the ligand binding domain of the oestrogen receptor (AsiSI-ER)2,3, together with a genome wide chromatin immunoprecipitation-sequencing (ChIP-seq) approach, we establish that distinct DSBs induced across the genome are not necessarily repaired by the same pathway. Indeed, we identify an HR-prone subset of DSBs that recruit the HR protein RAD51, undergo resection, and rely on RAD51 for efficient repair. These DSBs are located in actively transcribed genes, and repair at such DSBs can be switched to RAD51-independent repair pathway upon transcriptional inhibition. Moreover, we show that HR is targeted to transcribed loci thanks to the elongation-associated H3K36me3 histone mark. Indeed depletion of HYPB, the main H3K36 tri methyltransferase severally impedes the use of HR at those DSBs. Our study, thereby demonstrates a clear role for chromatin in DSB repair pathway choice in human cells.
2014-03-23 | E-MTAB-1241 | biostudies-arrayexpress
Project description:Every chromosome requires at least one crossover to be faithfully segregated during meiosis. At least two levels of regulation govern crossover distribution; where the initiating DNA double-strand breaks (DSBs) occur and whether those DSBs are repaired as crossovers. We mapped meiotic DSBs in budding yeast by identifying sites of DSB-associated single-stranded DNA (ssDNA) accumulation. These analyses revealed substantial DSB activity in regions close to centromeres, where crossover formation is largely absent. Our data suggest that centromeric suppression of recombination occurs at the level of break repair rather than DSB formation. Additionally, we found an enrichment of DSBs within a ~100-kb region near the ends of all chromosomes. Introduction of new telomeres was sufficient to induce large ectopic regions of increased DSB formation, revealing a remarkable long-range effect of telomeres on DSB formation. The concentration of DSBs close to chromosome ends increases the relative DSB density on small chromosomes, providing an interference-independent mechanism to ensure that all chromosomes receive at least one crossover per homolog pair. Together, our results indicate that selective DSB repair accounts for crossover suppression near centromeres, and suggest a simple telomere-guided mechanism to ensure sufficient DSB activity on all chromosomes. Keywords: ssDNA analysis, comparative genomic hybridization, ChIP-chip
2007-11-15 | GSE9503 | GEO
Project description:DNA double-strand break (DSB) repair by homologous recombination is confined to the S and G2 phases of the cell cycle partly due to 53BP1 antagonizing DNA end resection in G1 phase and non-cycling quiescent (G0) cells where DSBs must be repaired by non-homologous end joining (NHEJ). Unexpectedly, we uncovered extensive MRE11- and CtIP-dependent DNA end resection at DSBs in G0 mammalian cells. A whole genome CRISPR/Cas9 screen revealed the DNA-dependent kinase (DNA-PK) complex as a key factor in promoting DNA end resection in G0 cells. In agreement, depletion of FBXL12, which promotes ubiquitylation and removal of the KU70/KU80 subunits of DNA-PK from DSBs, promotes even more extensive resection in G0 cells. In contrast, a requirement for DNA-PK in promoting DNA end resection in cycling cells at the G1 or G2 phase cells was not observed. Our findings establish that DNA-PK uniquely promotes DNA end resection in G0, but not in G1 or G2 phase cells, and has important implications for DNA DSB repair in quiescent cells.
2022-05-16 | GSE186086 | GEO
Project description:DNA double-strand break (DSB) repair by homologous recombination is confined to the S and G2 phases of the cell cycle partly due to 53BP1 antagonizing DNA end resection in G1 phase and non-cycling quiescent (G0) cells where DSBs must be repaired by non-homologous end joining (NHEJ). Unexpectedly, we uncovered extensive MRE11- and CtIP-dependent DNA end resection at DSBs in G0 mammalian cells. A whole genome CRISPR/Cas9 screen revealed the DNA-dependent kinase (DNA-PK) complex as a key factor in promoting DNA end resection in G0 cells. In agreement, depletion of FBXL12, which promotes ubiquitylation and removal of the KU70/KU80 subunits of DNA-PK from DSBs, promotes even more extensive resection in G0 cells. In contrast, a requirement for DNA-PK in promoting DNA end resection in cycling cells at the G1 or G2 phase cells was not observed. Our findings establish that DNA-PK uniquely promotes DNA end resection in G0, but not in G1 or G2 phase cells, and has important implications for DNA DSB repair in quiescent cells.
2022-05-16 | GSE186085 | GEO
Project description:DNA double-strand breaks (DSBs) can be repaired by several pathways. In eukaryotes, repair pathway choice – the cellular decision making underlying DSB repair – occurs at the level of DSB resection and is controlled by the cell cycle. Upon cell cycle-dependent activation, cyclin-dependent kinases (CDKs) phosphorylate resection proteins and thereby stimulate DSB resection and repair by homologous recombination (HR). We uncovered a novel role for the Dbf4-dependent kinase Cdc7 (DDK) in regulating HR and DNA end resection. We therefore analyzed phosphorylation substrates of DDK by phosphoproteomics, where we compared wild type, bob1-1 and bob1-1dbf4∆ cells in M-phase arrested S.cerevisiae.
2024-02-15 | PXD042607 | Pride
Project description:Non-homologous end-joining (NHEJ) plays an important role in double-strand break (DSB) repair of DNA. Recent studies have shown that the error patterns of NHEJ are strongly biased by sequence context, but these studies were based on relatively few templates. To investigate this more thoroughly, we systematically profiled ~1.16 million independent mutational events resulting from CRISPR/Cas9-mediated cleavage and NHEJ-mediated DSB repair of 6,872 synthetic target sequences, introduced into a human cell line via lentiviral infection. We find that: 1) insertions are dominated by 1 bp events templated by sequence immediately upstream of the cleavage site, 2) deletions are predominantly associated with microhomology, and 3) targets exhibit variable but reproducible diversity with respect to the number and relative frequency of the mutational outcomes to which they give rise. From these data, we trained a model (Lindel) that uses local sequence context to predict the distribution of mutational outcomes. Exploiting the bias of NHEJ outcomes towards microhomology mediated events, we demonstrate the programming of deletion patterns by introducing microhomology to specific locations in the vicinity of the DSB site. We anticipate that our results will inform investigations of DSB repair mechanisms as well as the design of CRISPR/Cas9 experiments for diverse applications including genome-wide screens, gene therapy, lineage tracing and molecular recording.
2019-06-07 | GSE131421 | GEO
Project description:Translocations, that occur when two DNA Double Strand breaks (DSBs) are abnormally rejoined, represent highly deleterious genome rearrangements favoring cancer apparition and progression. However, the mechanisms that drive their formation are yet poorly deciphered. One prerequisite for translocation is the juxtaposition of two distant DSBs, an event that would be favored if DSB cluster, i.e. are brought together in close spatial proximity within the nucleus. Whether DSB cluster in higher eukaryotes has been subjected to a strong controversy over the past decade, due to conflicting results obtained using microscopy based methods1-9. Here we used for the first time a high throughput chromosome conformation capture assay (Capture Hi-C10) to investigate DSB clustering. We unambiguously found that DSBs do cluster in human nuclei but only when induced in transcriptionally active genes. Clustering of damaged genes mainly occur during the G1 cell cycle phase and coincide with delayed repair. Moreover DSB clustering depends on the MRN complex, as well as the Formin 2 (FMN2) nuclear actin organizer and the LINC (LInker of Nuclear and Cytoplasmic skeleton) complex, suggesting that active mechanisms promote DSB clustering. This work reveals that when damaged, active genes exhibit a very peculiar behavior compared to the rest of the genome, being mostly left unrepaired and clustered in G1 while being repaired by homologous recombination in post-replicative cells.
2017-04-19 | E-MTAB-4846 | biostudies-arrayexpress