Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE57283 | GEO | 2014/11/29
SECONDARY ACCESSION(S): PRJNA246166
REPOSITORIES: GEO
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Project description:We identified robust cleavage by non-canonically-targeted SpCas9 that target protospacer with a few bases from the canonical NGG PAM in biochemistry assay. To determine whether this spaced SpCas9 cleavage exists in human cells, we employed high-throughput genome-wide translocation sequencing (LAM-HTGTS) using SpCas9 with three independent canonical sgRNAs served as bait (bait-A, bait-L and bait-D) to compared the cleavage activities of canonically-targeted and non-canonically-targeted SpCas9 in eight locus (RAG1B-D, RAG1K-O) in chromosome 11. We found that almost all 1bp-target shifted SpCas9 generated substantial DSBs as the canonical controls. While detected less frequently, 2bp-targeted shifted SpCas9 generated significant amounts of DSBs in RAG1D and RAG1L locus. These findings underscore the spaced PAM-mediated DSBs occurred in living cells.
2022-02-28 | GSE192459 | GEO
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:We recently discovered 27 recurrent DNA double-strand break (DSB) clusters (RDCs) in mouse neural stem/progenitor cells (NSPCs). Most RDCs occurred across long, late-replicating RDC genes and were found only after mild inhibition of DNA replication. RDC genes share intriguing characteristics, including encoding surface proteins that organize brain architecture and neuronal junctions, and are genetically implicated in neuropsychiatric disorders and/or cancers. RDC identification relies on high-throughput genome-wide translocation sequencing (HTGTS), which maps recurrent DSBs based on their translocation to "bait" DSBs in specific chromosomal locations. Cellular heterogeneity in 3D genome organization allowed unequivocal identification of RDCs on 14 different chromosomes using HTGTS baits on three mouse chromosomes. Additional candidate RDCs were also implicated, however, suggesting that some RDCs were missed. To more completely identify RDCs, we exploited our finding that joining of two DSBs occurs more frequently if they lie on the same cis chromosome. Thus, we used CRISPR/Cas9 to introduce specific DSBs into each mouse chromosome in NSPCs that were used as bait for HTGTS libraries. This analysis confirmed all 27 previously identified RDCs and identified many new ones. NSPC RDCs fall into three groups based on length, organization, transcription level, and replication timing of genes within them. While mostly less robust, the largest group of newly defined RDCs share many intriguing characteristics with the original 27. Our findings also revealed RDCs in NSPCs in the absence of induced replication stress, and support the idea that the latter treatment augments an already active endogenous process.
2018-01-19 | GSE106822 | GEO
Project description:Genome editing typically involves recombination between donor nucleic acids and genomic sequences subjected to double-stranded DNA breaks (DSBs) made by programmable nucleases (e.g. CRISPR-Cas9). Yet, amongst other deleterious by-products, DSBs yield translocations, off-target mutations and, most pervasively, unpredictable on-target allelic disruptions. Remarkably, hitherto, the untoward phenotypic consequences of on-target disruptions at allelic and non-allelic (e.g. pseudogene) sequences have received scant scrutiny and, crucially, remain to be addressed. Here, we demonstrate that gene-edited cells can lose fitness due to on-target DSBs and report that simultaneous single-stranded DNA break formation at donor and target DNA by CRISPR-Cas9 “nickases” overcomes, to a great extent, such genotype-phenotype disrupting events. Moreover, in trans paired nicking gene editing can efficiently and precisely add large DNA segments (i.e. live-cell reporter tags) into essential and multiple-copy genomic sequences while circumventing most of the allelic and non-allelic collateral mutations and chromosomal rearrangements characteristic of nuclease-dependent gene editing procedures.
2019-12-04 | GSE135064 | GEO
Project description:High-throughput genome-wide translocation sequencing (HTGTS) is a robust approach to identify genome-wide translocation junctions. We performed HTGTS to study the fate of introduced c-myc DSBs in mouse splenic B cells activated for activation cytidine deaminase (AID)-dependent class switch recombination (CSR). We found frequent translocations of c-myc DSBs to AID-initiated DSBs in IgH switch regions in wild-type (WT) and ATM-deficient B cells. However, c-myc also translocated frequently to newly generated DSBs within a 35-megabase region downstream of IgH in ATM-deficient, but not WT, CSR-activated B cells. Moreover, we found such DSBs and translocations in activated B cells that did not express AID or undergo CSR. These findings indicate that ATM deficiency leads to formation of chromosome 12 dicentrics via RAG-initiated IgH DSBs in progenitor B cells and that these dicentrics can be propagated developmentally into mature B cells where they generate new DSBs downstream of IgH via breakage-fusion-bridge cycles. Preparation of libraries from WT or ATM-deficient activated by a-CD40/IL4 or RP105.
2014-06-18 | E-GEOD-58599 | biostudies-arrayexpress
Project description:High-throughput genome-wide translocation sequencing (HTGTS) is a robust approach to identify genome-wide translocation junctions. We performed HTGTS to study the fate of introduced c-myc DSBs in mouse splenic B cells activated for activation cytidine deaminase (AID)-dependent class switch recombination (CSR). We found frequent translocations of c-myc DSBs to AID-initiated DSBs in IgH switch regions in wild-type (WT) and ATM-deficient B cells. However, c-myc also translocated frequently to newly generated DSBs within a 35-megabase region downstream of IgH in ATM-deficient, but not WT, CSR-activated B cells. Moreover, we found such DSBs and translocations in activated B cells that did not express AID or undergo CSR. These findings indicate that ATM deficiency leads to formation of chromosome 12 dicentrics via RAG-initiated IgH DSBs in progenitor B cells and that these dicentrics can be propagated developmentally into mature B cells where they generate new DSBs downstream of IgH via breakage-fusion-bridge cycles.
2014-06-18 | GSE58599 | GEO
Project description:Our laboratory identified robust recurrent DNA double-strand break (DSB) cluster (“RDC”) genes in mouse neural stem/progenitor cells (NSPCs) by applying the high-throughput, genome-wide, translocation sequencing (HTGTS) method. Genomic alterations of most of the identified RDC-genes have been associated with psychiatric disorders such as autism and schizophrenia and several are altered in brain cancer. The most robust mouse RDCs are all in genes that tend to be very long, actively transcribed and were enhanced after mild inhibition of replication stress. The common transcription and replication characteristics we observe in RDC-genes suggest that frequent RDC DSBs might be generated by collision between transcription and replication processes. However, the underlying mechanism of RDC formation is still unknown. To elucidate mechanisms that generate and resolve DSBs in these cells, we established an in vitro system of induced neural progenitor cells derived from embryonic stem cells. HTGTS bait-DSBs introduced by CRISPR/Cas9 on three mouse chromosomes identified only 5 RDC-genes in embryonic stem cells and 27 RDC-genes in the neural progenitor cells differentiated from them. All RDC-genes identified in induced neural progenitor cells belonged to the group of genes identified in primary neural and progenitor cells. These results indicate that our in vitro differentiation system is both effective and robust in terms of recapitulating our previous findings and facilitating mechanistic studies.
2020-01-13 | GSE142289 | GEO
Project description:Our laboratory identified robust recurrent DNA double-strand break (DSB) cluster (“RDC”) genes in mouse neural stem/progenitor cells (NSPCs) by applying the high-throughput, genome-wide, translocation sequencing (HTGTS) method. Genomic alterations of most of the identified RDC-genes have been associated with psychiatric disorders such as autism and schizophrenia and several are altered in brain cancer. The most robust mouse RDCs are all in genes that tend to be very long, actively transcribed and were enhanced after mild inhibition of replication stress. The common transcription and replication characteristics we observe in RDC-genes suggest that frequent RDC DSBs might be generated by collision between transcription and replication processes. However, the underlying mechanism of RDC formation is still unknown. To elucidate mechanisms that generate and resolve DSBs in these cells, we established an in vitro system of induced neural progenitor cells derived from embryonic stem cells. HTGTS bait-DSBs introduced by CRISPR/Cas9 on three mouse chromosomes identified only 5 RDC-genes in embryonic stem cells and 27 RDC-genes in the neural progenitor cells differentiated from them. All RDC-genes identified in induced neural progenitor cells belonged to the group of genes identified in primary neural and progenitor cells. These results indicate that our in vitro differentiation system is both effective and robust in terms of recapitulating our previous findings and facilitating mechanistic studies.
2020-01-13 | GSE142312 | GEO
Project description:RAG initiates V(D)J recombination in developing lymphocytes by generating "on-target" DNA double-stranded breaks at bona fide recombination signal sequence (RSS) pairs. We now employ RAG-generated DNA breaks in endogenous or ectopically-inserted RSS pairs as bait to identify huge numbers of RAG "off-target" sites. These off-target DNA breaks occur across convergent CTCF-binding element (CBE)-flanked loop domains containing the bait RSS pairs. Such off-target cleavage occurs at the simple CAC motif that defines the RSS cleavage-site and shows orientation-dependence best explained by a two-dimensional tracking mechanism. Deletion of the CBE-based IGCR1 IgH regulatory element disrupts antibody IgH recombination domains and, correspondingly, alters distributions of RAG on- and off-targets across the IgH locus. RAG off-targets frequently involved in chromosomal translocations occur in convergent RSS pairs at enhancer regions within a loop. Our findings reveal how RAG is developmentally focused and re-focused in lymphocytes and implicate mechanisms by which chromatin domains harness biological processes within them. We performed high-throughput genome-wide translocation sequencing (HTGTS) in progenitor B cells or cell lines to study signatures of RAG on and off-targeting activity using RAG-generated breaks in endogenous or ectopically-inserted paired bona fide RSSs as bait. Sequenceing was dong by Illumina Miseq.
2015-09-15 | E-GEOD-73007 | biostudies-arrayexpress
Project description:During B cell development, RAG endonuclease cleaves immunoglobulin heavy chain (IgH) V, D, and J gene segments and orchestrates their fusion as deletional events that assemble a V(D)J exon in the same transcriptional orientation as adjacent Cμ constant region exons. In mice, six additional sets of constant region exons (CHs) lie 100-200kb downstream in the same transcriptional orientation as V(D)J and Cμ exons. Long repetitive switch (S) regions precede Cμ and downstream CHs. In mature B cells, class switch recombination (CSR) generates different antibody classes by replacing Cμ with a downstream CH. Activation Induced Cytidine Deaminase (AID) initiates CSR by promoting deamination lesions within Sμ and a downstream acceptor S region; these lesions are converted into DNA double strand breaks (DSBs) by general DNA repair factors. Productive CSR must occur in a deletional orientation by joining the upstream end of an Sμ DSB to the downstream end of an acceptor S region DSB. However, the relative frequency of deletional to inversional CSR junctions had not been measured. Thus, whether orientation-specific joining is a programmed mechanistic feature of CSR as it is for V(D)J recombination and, if so, how this is achieved was unknown. To address this question, we adapted high throughput genome wide translocation sequencing (HTGTS) into a highly sensitive DSB end-joining assay and applied it to endogenous AID-initiated S region DSBs. We find that CSR indeed is programmed to occur in a productive deletional orientation and does so via an unprecedented mechanism that involves in cis IgH organizational features in combination with frequent S region DSBs initiated by AID. We further implicate ATM-dependent DSB response (DSBR) factors in enforcing this mechanism and provide a solution to the enigma of why CSR is so reliant on the 53BP1 DSBR factor. We performed high-throughput genome-wide translocation sequencing (HTGTS) with different B cell genotypes that induces either I-SceI or AID-initiated DSBs as bait to study their joining pattern to AID-initiated S region breaks Please note that the 'ΔSγ1_2xI/ΔSµ_2xI-3' raw data files were analyzed twice with different reference assemblies to address specific points, and associated with both 'ΔSγ1_2xI_ΔSµ_2xI-3'.txt and Sγ1_2xI_ΔSµ_2xI-3'_trans-Sm.txt processed data files. As for the reference genome mm9_129_IgHC, it is a custom built generated based on mm9 (Bl6 line based mouse genome) and the partial genome sequence of another mouse line 129sv. It is not currently available in any public database however can be easily obtained either from the authors or self-built using information provided in the associated manuscript.
2015-08-26 | E-GEOD-71005 | biostudies-arrayexpress