Massively parallel profiling and predictive modeling of the outcomes of CRISPR/Cas9-mediated double-strand break repair
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE131421 | GEO | 2019/06/07
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:DNA-PKcs is a crucial component of the non-homologous end joining (NHEJ) repair machinery. To investigate its function in human cell lines, we conducted a study using K562 and HEK293T cell lines. We introduced twinned DNA double-strand breaks (DSBs) or genome-wide DSBs into these cell lines via nucleofection and transfection, respectively. Subsequently, we employed high-throughput genome translocation sequencing (HTGTS) to capture the translocation events (i.e., ligation between "prey(s)" and "bait") and the rejoining events (i.e., direct repair within the "bait" locus) under different conditions, including with or without DNA-PKcs inhibition or deletion. We quantified the number of translocation events by normalizing them to the number of rejoining events, denoted as TL. Interestingly, DNA-PKcs inhibition led to an increase in TL, indicating a higher frequency of translocations. However, it is important to note that chromosomal translocations still predominantly relied on NHEJ despite DNA-PKcs inhibition. Furthermore, we observed that DNA-PKcs deletion resulted in an elevated utilization of microhomology in translocation formation. Nevertheless, NHEJ remained the primary mechanism driving translocation events.These findings provide valuable insights into the role of DNA-PKcs in the repair pathways involved in translocation events in human cell lines. The utilization of HTGTS allowed us to comprehensively analyze the effects of DNA-PKcs inhibition and deletion, shedding light on the interplay between NHEJ and alternative repair mechanisms in translocation formation.
2023-09-01 | GSE232940 | GEO
Project description:The proposal aims to characterise the pathways of DSB repair and recombination in Arabidopsis with the main focus of this research being the NHEJ pathway of illegitimate recombination. We will build on our expertise and resources in the field of DSB repair in plants, using the Arabidopsis NHEJ mutant atku80 which is an excellent model system for the study of DSB repair in higher eukaryotes (West et al., 2002 Plant J. 31, 517-28). Comparison of the transcriptome in NHEJ mutant and wild type plants under different experimental conditions will identify novel candidate genes involved in DNA DSB repair or damage signalling pathways.We will conduct 4 separate experiments on Arabidopsis seedlings grown on 0.5 MS media: the first will be a control consisting of Wassilewskija (WS-2) plants grown under standard conditions. In the second experiment WS plants will be exposed to the chemotherapeutic agent bleomycin (1 microgram per ml). This agent has been shown to cause both single and double strand breaks in the genome of Arabidopsis seedlings (Menke et al., 2001 Mut. Res. 493, 87-93). This agent is used in preference to gamma irradiation, which causes high levels of oxidative damage to cellular components. These experiments will characterise for the first time the transcriptional responses of Arabidopsis cells to the specific induction of DNA strand breaks. The transcript profile will also be determined in untreated atku80 mutants. This experiment will identify the components of novel and previously characterised DNA repair pathways up regulated in the mutant background. Finally, transcript analysis of bleomycin treated atku80 mutants and comparison with untreated mutant plants and both untreated and treated WS plants will identify novel putative DSB repair genes up regulated in response to genotoxic stress in the absence of a Ku80 mediated DSB repair pathway.The results of these studies will provide new and important information which will be instrumental in identifying novel genes and pathways involved in DNA repair and genome stability in plants and help elucidate the fundamental differences that exist between animal and plant DSB repair processes. Experiment Overall Design: Number of plants pooled:20
2008-06-14 | E-GEOD-6178 | biostudies-arrayexpress
Project description:Classical non-homologous end-joining (C-NHEJ) is a major mammalian DNA double strand break (DSB) repair pathway. Core C-NHEJ factors, such as XRCC4, are required for joining DSB intermediates of the G1 phase-specific V(D)J recombination reaction in progenitor lymphocytes. Core factors also contribute to joining DSBs in cycling mature B-lineage cells, including DSBs generated during antibody class switch recombination (CSR) and DSBs generated by ionizing radiation (IR). The XLF C-NHEJ factor is dispensable for V(D)J recombination in normal cells, but, due to functional redundancy, is absolutely required for this process in cells deficient for the ATM DSB response factor. The recently identified PAralogue of XRCC4 and XLF (PAXX) factor has homology to these two proteins and variably contributes to IR-induced DSB repair in human and chicken cells. We now report that PAXX is dispensable for joining V(D)J recombination DSBs in G1-arrested mouse pro-B cell lines, dispensable for joining CSR-associated DSBs in a cycling mouse B cell line, and dispensable for normal IR-resistance in both G1-arrested and cycling pro-B lines. However, we find that combined deficiency for PAXX and XLF in G1-arrested pro-B lines abrogates DSB joining during V(D)J recombination and sensitizes the cells to IR exposure. Thus, PAXX provides core C-NHEJ factor-associated functions in the absence of XLF and vice versa in G1-arrested Pro-B cell lines. Finally, we also find that PAXX-deficiency has no impact on V(D)J recombination DSB joining in ATM-deficient pro-B lines. We discuss implications of these findings with respect to potential PAXX and XLF functions in C-NHEJ. Examination of CSR Switch mu-to-alpha junctions from mu bait DSBs using LAM-HTGTS and Illumina Miseq. Two clones of PAXX-/- and XLF-/- and 1 clone of Ligase4-/- were derived from the parental CH12F3 line; the second Ligase4-/- clone was acquired from Keifei Yu. Two clones of XLF-/-PAXX-/-CH12 cells were derived from one of the PAXX-/- clones. Three biological replicates were analyzed for each clone.
2016-08-19 | E-GEOD-84102 | biostudies-arrayexpress
Project description:Cell cycle is a major determinant of DNA double-strand break (DSB) repair pathway choice with homologous recombination (HR) that is most active in S phase cells and non-homologous end-joining (NHEJ) that dominates in G1 phase cells. A third less well-defined mechanism, 'alternative end-joining', has been shown to promote error-prone repair in NHEJ- or HR-deficient cells. Here, we have used a physiologic system of NHEJ-mediated genomic rearrangements induced by the site-specific RAG1/2 endonuclease in G1 cells to investigate the fate of unrepaired G1 DSBs upon entry into the cell cycle. We show that, in the absence of XRCC4, alternative end-joining rescues RAG-induced DSB repair and promotes chromosome translocations upon G1 cell cycle exit.
2020-08-24 | GSE138136 | GEO
Project description:Cell cycle is a major determinant of DNA double-strand break (DSB) repair pathway choice with homologous recombination (HR) that is most active in S phase cells and non-homologous end-joining (NHEJ) that dominates in G1 phase cells. A third less well-defined mechanism, 'alternative end-joining', has been shown to promote error-prone repair in NHEJ- or HR-deficient cells. Here, we have used a physiologic system of NHEJ-mediated genomic rearrangements induced by the site-specific RAG1/2 endonuclease in G1 cells to investigate the fate of unrepaired G1 DSBs upon entry into the cell cycle. We show that, in the absence of XRCC4, alternative end-joining rescues RAG-induced DSB repair and promotes chromosome translocations upon G1 cell cycle exit.
2020-08-24 | GSE138135 | GEO
Project description:Formation of templated insertions at DNA double-strand breaks (DSBs) is very common in cancer cells. The mechanisms and enzymes regulating these events are largely unknown. Here, we investigated templated insertions in yeast at DSBs using amplicon sequencing across a repaired locus. We document very short (most ~5-34 bp), templated inverted duplications at DSBs. They are generated through a foldback mechanism that utilizes microhomologies adjacent to the DSB. Enzymatic requirements suggest a hybrid mechanism wherein one end requires Polδ-mediated synthesis while the other end is captured by nonhomologous end joining (NHEJ). This process is exacerbated in mutants with low levels or mutated RPA (rtt105Δ; rfa1-t33) or extensive resection mutant (sgs1Δ exo1Δ). Templated insertions from various distant genomic locations also increase in these mutants as well as in rad27Δ and originate from fragile regions of the genome. Among complex events, common events are insertions of two sequences, originating from the same locus and with inverted orientation. Evidence suggests that inversions are also formed by microhomology-mediated template switching. Taken together, our results suggest that limited RPA, typical in many cancers, may contribute to the high level of microhomology-mediated templated insertions.
2024-03-07 | GSE260753 | GEO
Project description:Meiotic recombination facilitates accurate pairing and faithful segregation of homologous chromosomes by forming physical connections (crossovers) between homologs. Developmentally programmed DNA double-strand breaks (DSBs) generated by Spo11 protein (Rec12 in fission yeast) initiate meiotic recombination. Until recently, attempts to address the basis of the highly non-random distribution of DSBs on a genome-wide scale have been limited to 0.1M-bM-^@M-^S1 kb resolution of DSB position. We have assessed individual DSB events across the Schizosaccharomyces pombe genome at near-nucleotide resolution by deep-sequencing the short oligonucleotides connected to Rec12 following DNA cleavage. The single oligonucleotide size-class generated by Rec12 allowed us to effectively analyze all break events. Our high-resolution DSB map shows that the influence of underlying nucleotide sequence and chromosomal architecture differs in multiple ways from that in budding yeast. Rec12 action is not strongly restricted to nucleosome-depleted regions but is nevertheless spatially biased with respect to chromatin structure. Furthermore, we find strong evidence across the genome for differential DSB repair previously predicted to account for crossover invariance (constant cM/kb in spite of DSB hotspots). Our genome-wide analyses demonstrate evolutionarily fluid factors contributing to crossover initiation and its regulation. Three samples total: one sample sequenced by 454 and two technical replicates (independent adaptor ligations from material purified from one culture) sequenced by SOLiD
2013-08-20 | E-GEOD-49977 | biostudies-arrayexpress
Project description:Large scale analysis of balanced chromosomal translocation breakpoints has shown nonhomologous end joining and microhomology-mediated repair to be the main drivers of interchromosomal structural aberrations. Breakpoint sequences of de novo unbalanced translocations have not yet been investigated systematically. We analyzed 12 de novo translocations and mapped the breakpoints in 9. Surprisingly, in contrast to balanced translocations, we identify non-allelic homologous recombination (NAHR) between (retro)transposable elements and especially long interspersed elements (LINEs) as the main mutational mechanism. This finding implicates (retro)transposons to be a major driver of genomic rearrangements and exposes a profoundly different mutational mechanism compared to balanced chromosomal translocations. Furthermore, we show the existence of compound maternal/paternal derivative chromosomes, reinforcing the hypothesis that human cleavage stage embryogenesis is a cradle of chromosomal rearrangements. In total 36 non-amplified genomic DNA samples (12 patients plus parents) extracted from blood or amniocytes were analyzed by 250K Nsp I SNP arrays (GEO accession number GPL3718).
2012-07-12 | E-GEOD-39303 | biostudies-arrayexpress