BACKGROUND:G-quadruplex (G4) structures are secondary structures that can form in guanine-rich single stranded DNA sequences. These play important roles in biological processes such as regulation of gene expression but can also pose challenges to DNA replication and lead to genome instability. The SMARCA4 (BRG1) subunit of the SWI/SNF chromatin remodelling complexes has been identified as a G4 binding protein, and evidence suggests that this interaction can promote SWI/SNF-dependent gene expression. SMARCA4 is frequently misregulated in cancer, where genome instability is common, but whether there is an impact of SMARCA4 on G4 stability was not known. RESULTS:Here, we show that SMARCA4 prevents genome instability at G4s. Mapping unrepaired DNA breaks reveals that these preferentially co-localise with G4 forming structures in SMARCA4-deficient cells. Moreover, using whole genome sequencing approaches, we find that misrepair events in SMARCA4-deficient cells are more likely to map to G4 forming sequences. Consistent with this, SMARCA4-deficient cells show sensitivity to the G4 ligand pyridostatin and defective pyridostatin-induced DNA damage responses. Notably, analysis of cancer patient data shows that SMARCA4-deficient samples have an increased proportion of G4-associated mutations when compared with SMARCA4-proficient samples. CONCLUSIONS:These findings suggest that SMARCA4 plays a crucial role in maintaining stability at G4 motifs. This insight provides valuable information about the functional significance of G4 structures and their interaction with SMARCA4, particularly in the context of cancer.

Background/Objectives: To date, no validated scoring system can accurately predict the responses of acute myeloid leukaemia (AML) patients to induction chemotherapy (CTX). Current risk assessment relies on complex cytogenetic and molecular abnormalities and focuses on mutations in genes considered fundamental to leukaemogenesis. Methods: We performed bioinformatic analysis of targeted sequencing (TS) data from 111 genes in 1552 AML patients, focusing on mutational patterns derived from single-nucleotide variant (SNV) catalogues. The SNV catalogues were analysed using non-negative matrix factorisation (NNMF), a linear dimensionality-reduction approach, to extract risk-defining recursive signatures (RSs) and to distinguish responders from resistant patients following induction CTX. To enable patient-level prediction, we complemented NNMF with a Random Forest (RF) model. Given the class imbalance between responders and resistant cases, model performance was improved by applying the Synthetic Minority Over-sampling Technique (SMOTE) and by incorporating germline variants alongside somatic mutations. Results: NNMF-derived RSs captured clinically relevant structures in patients’ mutational profiles and clustered patients by treatment response, indicating that the diagnostic targeted sequencing data contain sufficient information for risk stratification and treatment response prediction. At the single-patient level, RF models incorporating balanced data and germline variation improved predictive performance compared with unbalanced somatic-only models. Conclusions: These findings demonstrate that machine learning applied to targeted sequencing data can extract clinically informative mutational structures and improve risk stratification in AML, supporting its potential integration into precision treatment decision-making.

An intronic hexanucleotide repeat expansion in C9orf72 is the leading genetic cause of both frontotemporal dementia and amyotrophic lateral sclerosis (C9-FTD/ALS). We have previously demonstrated that CRISPR/Cas9 excision of the repeat expansion in patient iPSCs reverts pathological hallmarks of C9-FTD/ALS. Here we aim to identify efficient and safe gRNAs for CRISPR spCas9 dual-gRNA excision of the C9-repeat expansion. Utilizing novel ddPCR and single-molecule sequencing assays, we screened 120 gRNA pairs, comparing 64 bi-allelic, intronic excisions of the repeat region to 56 allele-specific excisions of the mutant allele in patient iPSCs, ranking them by efficiency. Bi-allelic excisions of the intronic repeat region were more efficient than excisions of the mutant allele. Single gRNA indel rates can nominate likely efficient gRNA pairs, but these pairs must be tested empirically. The length of the repeat expansion did not impact excision efficiency; rather, the activity of individual gRNAs drove excision efficiencies. Using whole genome sequencing and INDUCE-seq, we found only one detectable off-target of those nominated by Cas-OFFinder and CHANGE-seq across 4 of the most efficient gRNAs. This study advances the development of targeted therapies for C9-FTD/ALS and establishes a framework for dual gRNA screening in patient iPSCs applicable to other repeat expansions.

Background : Signaling by estrogen-receptor alpha (ERα) plays a major role in breast cancer initiation and ERα-mediated DNA damage has been implicated in development of resistance to endocrine therapies. Investigations of the mechanism of DNA damage mediated by ERα signaling are carried out in breast cancer cell lines due to the lack of ERα+ normal human breast epithelial cells lines (HBEC). Defining the mechanisms by which ERα induces DNA damage and initiates tumorigenesis requires normal HBECs that express ERα, demonstrate estrogenic responses, and are amenable to long term propagation in culture. Methods : We utilized lentiviral expression of an inducible ERα construct to generate four HBEC lines (HBEC- ESR1 ). We studied these cells for ERα-dependent responses using a luciferase reporter, endogenous gene expression and proliferation assays. RNA-Seq was performed to characterize the ERα-mediated transcriptomic patterns in the four HBEC lines. ERα mediated DNA double strand breaks (DSBs) were analyzed using γH2AX immunofluorescence. Results : Expression and functional activation of ERα were observed in all HBEC- ESR1 lines, whereas proliferation in response to 17β-estradiol (E2) was observed in 3 of the cell lines. Proliferative responses were due to intrinsic signaling within the HBECs as conditioned media from the cells failed to cause proliferation. A total of 682 genes were differentially expressed at 24h following treatment with 10nM E2 with 43% of these genes were also observed in ERα+ breast cancer cell lines (MCF7 or T47D). Gene-set enrichment analysis identified differential expression of genes in ERα signaling pathways and DNA repair pathways in E2-treated cells. E2-induced ERα signaling also increased γH2AX foci in 3 of the 4 cell lines. Levels of DSBs were increased by inhibition of the non-homologous end-joining (NHEJ) and homologous recombination (HR) pathways. DSBs were also increased in MCF10A- ESR1 cells heterozygous for the BRCA1185delAG mutation causing a truncated protein. Conclusions : Inducible expression of ERα in immortalized HBECs recapitulate transcriptional, replicative and DNA damage responses. Increased DSBs in MCF10A- ESR1 cells with heterozygous mutation of BRCA1 indicate haploinsufficiency and the potential for increased genetic instability due to ERα signaling. ### Competing Interest Statement The authors have declared no competing interest.

Background & Aim Advances in genome editing enable the development of innovative cell and gene therapies. However, gene editing introduces risks, including malignant transformation of target cells due to ex vivo culturing, which can result in mutations conferring growth advantages. Direct genomic instability and selection for oncogenic mutations in cells with pre-existing DNA repair defects may also occur.Additionally, off-target effects from gene editing tools can cause unintended DNA breaks, activating proto-oncogenes or disrupting tumor suppressors, potentially driving carcinogenesis. Off-target mutagenesis may lead to neoantigens, triggering autoimmunity or other dysfunctions. Rare penetrant mutations further emphasize the importance of monitoring off-target effects to ensure safety.Standardized assays for assessing the safety of gene editing therapies are currently lacking. This study introduces INDUCE-seq, a method for detecting genome breaks at targeted and off-target sites. Methodology CRISPR-Cas9 editing of five genetic targets was performed by two industry partners on two cell types. INDUCE-seq measured genome breaks, while Duplex Sequencing detected rare mutations at these sites with high sensitivity. CRISPR-Cas9-based gene editing of five well-studied genetic targets was conducted by two independent industry partners (AZ & Novartis), using two different cell types. On- and off-target gene editing was assessed by measuring breaks in the genome using INDUCE-seq. The genetic changes at these locations were subsequently measured using error-corrected sequencing using Duplex-seq4 which allows for sensitive detection of mutations. Results INDUCE-seq reproducibly detects on-target editing between the two laboratories and cell types. INDUCE-seq reproducibly identifies off-targets between laboratories. INDUCE-seq detects both common and unique off-targets in different cell types. DSB recurrency at both on and off-targets correlates with mutational frequency Conclusion This study introduces INDUCE-seq, a method for detecting genome breaks at targeted and off-target sites. CRISPR-Cas9 editing of five genetic targets was performed by two industry partners on two cell types. INDUCE-seq measured genome breaks, while Duplex Sequencing detected rare mutations at these sites with high sensitivity. Findings from this study aim to enhance the precision and safety of therapeutic gene editing.

DNA G-quadruplexes (G4s) are secondary structures with significant roles in regulating genome function and stability. Dysregulation of the dynamic formation of G4s is linked to genomic instability and disease, but the underlying mechanisms are not fully understood. In this study, we conducted a screen of chromatin-modifying enzymes and identified nine potential inhibitors of G4 formation, including seven that were not previously characterized. Among these, we highlight the role of BAZ2 chromatin remodelers as key suppressors of G4 DNA and G4-related genome instability. Depletion of BAZ2 subunits led to increased G4 formation, especially at transcriptional regulatory elements. BAZ2B was found to associate with G4 loci, suggesting that it plays a direct role in suppressing G4s. While BAZ2-deficient cells exhibited modest genomic instability, treatment with the G4-stabilizing ligand BRACO19 exacerbated double-strand breaks (DSBs), highlighting its utility as a tool to study G4-dependent genome instability. DSB profiling using INDUCE-seq uncovered distinct breakage patterns around G4s, further underscoring the impact of G4s on genome integrity. Notably, we found that within G4s, G repeats were more susceptible to DSBs than loops. These results establish BAZ2 chromatin remodeling complexes as direct regulators of G4 dynamics and provide new insights into G4-dependent genome instability. ### Competing Interest Statement The authors have declared no competing interest.

Regulators and industry are actively seeking improvements and alternatives to current models and approaches to evaluate potential carcinogenicity of gene therapies (GTs). A meeting of invited experts was organized by NC3Rs/UKEMS (London, March 2023) to discuss this topic. This article describes the consensus reached among delegates on the definition of vector genotoxicity, sources of uncertainty, suitable toxicological endpoints for genotoxic assessment of GTs, and future research needs. The collected recommendations should inform the further development of regulatory guidelines for the nonclinical toxicological assessment of GT products.

In recent years, various long non-coding RNAs (lncRNAs) involved in DNA damage response (DDR) have been identified and studied to deepen our understanding. However, there are rare reports on the association between lncRNAs and base excision repair (BER). Our designed DNA microarray identified dozens of functionally unknown lncRNAs, and their transcription levels significantly increased upon exposure to DNA damage inducers. One of them, named LIP (Long noncoding RNA Interacts with PARP-1), exhibited a significant alteration in transcription in response to methyl methanesulfonate (MMS) and temozolomide (TMZ) treatments. LIP knockdown or knockout cell lines are sensitive to MMS and TMZ, indicating that LIP plays a crucial role in DDR. The loss or insufficiency of LIP significantly influences the efficiency of BER in human cells, and it suggests that LIP participates in the BER pathway. The interaction between LIP and a key factor in BER, poly (ADP-ribose) polymerase 1 (PARP-1), has been confirmed. We identified and characterized LIP, a lncRNA, which is involved in DDR, significantly influences BER efficiency, and interacts with the BER key factor PARP-1. This advances our understanding of the connection between lncRNAs and BER, presenting the potential for the discovery of new drug targets.

Mammalian DNA replication relies on various DNA helicase and nuclease activities to ensure accurate genetic duplication, but how different helicase and nuclease activities are properly directed remains unclear. Here, we identify the ubiquitin-specific protease, USP50, as a chromatin-associated protein required to promote ongoing replication, fork restart, telomere maintenance, cellular survival following hydroxyurea or pyridostatin treatment, and suppression of DNA breaks near GC-rich sequences. We find that USP50 supports proper WRN-FEN1 localisation at or near stalled replication forks. Nascent DNA in cells lacking USP50 shows increased association of the DNA2 nuclease and RECQL4 and RECQL5 helicases and replication defects in cells lacking USP50, or FEN1 are driven by these proteins. Consequently, suppression of DNA2 or RECQL4/5 improves USP50-depleted cell resistance to agents inducing replicative stress and restores telomere stability. These data define an unexpected regulatory protein that promotes the balance of helicase and nuclease use at ongoing and stalled replication forks. Mammalian DNA replication relies on various helicases and nucleases to ensure accurate genetic duplication, but how these enzymes are properly directed is unclear. Here, the authors identify USP50 as a key protein for promoting ongoing replication, restarting stalled forks, maintaining telomeres, and ensuring cell survival.

The use of error-corrected Next Generation Sequencing (ecNG) to determine mutagenicity has been a subject of growing interest and potentially a disruptive technology that could supplement, and in time, replace current testing paradigms in preclinical safety assessment. Considering this, a Next Generation Sequencing Workshop was held at the Royal Society of Medicine in London in May 2022, supported by the United Kingdom Environmental Mutagen Society (UKEMS) and TwinStrand Biosciences (WA, USA), to discuss progress and future applications of this technology. In this meeting report, the invited speakers provide an overview of the Workshop topics covered and identify future directions for research. In the area of somatic mutagenesis, several speakers reviewed recent progress made with correlating ecNGS to classic in vivo transgenic rodent mutation assays as well as exploring the use of this technology directly in humans and animals, and in complex organoid models. Additionally, ecNGS has been used for detecting off-target effects of gene editing tools and emerging data suggest ecNGS potential to measure clonal expansion of cells carrying mutations in cancer driver genes as an early marker of carcinogenic potential and for direct human biomonitoring. As such, the workshop demonstrated the importance of raising awareness and support for advancing the science of ecNGS for mutagenesis, gene editing, and carcinogenesis research. Furthermore, the potential of this new technology to contribute to advances in drug and product development and improve safety assessment was extensively explored.

CRISPR-Cas9 is a powerful gene-editing technology; however, off-target activity remains an important consideration for therapeutic applications. We have previously shown that force-stretching DNA induces off-target activity and hypothesized that distortions of the DNA topology in vivo, such as negative DNA supercoiling, could reduce Cas9 specificity. Using single-molecule optical-tweezers, we demonstrate that negative supercoiling λ-DNA induces sequence-specific Cas9 off-target binding at multiple sites, even at low forces. Using an adapted CIRCLE-seq approach, we detect over 10,000 negative-supercoiling-induced Cas9 off-target double-strand breaks genome-wide caused by increased mismatch tolerance. We further demonstrate in vivo that directed local DNA distortion increases off-target activity in cells and that induced off-target events can be detected during Cas9 genome editing. These data demonstrate that Cas9 off-target activity is regulated by DNA topology in vitro and in vivo, suggesting that cellular processes, such as transcription and replication, could induce off-target activity at previously overlooked sites.

BACKGROUND:miR-346 was identified as an activator of Androgen Receptor (AR) signalling that associates with DNA damage response (DDR)-linked transcripts in prostate cancer (PC). We sought to delineate the impact of miR-346 on DNA damage, and its potential as a therapeutic agent. METHODS:RNA-IP, RNA-seq, RNA-ISH, DNA fibre assays, in vivo xenograft studies and bioinformatics approaches were used alongside a novel method for amplification-free, single nucleotide-resolution genome-wide mapping of DNA breaks (INDUCE-seq). RESULTS:miR-346 induces rapid and extensive DNA damage in PC cells - the first report of microRNA-induced DNA damage. Mechanistically, this is achieved through transcriptional hyperactivation, R-loop formation and replication stress, leading to checkpoint activation and cell cycle arrest. miR-346 also interacts with genome-protective lncRNA NORAD to disrupt its interaction with PUM2, leading to PUM2 stabilisation and its increased turnover of DNA damage response (DDR) transcripts. Confirming clinical relevance, NORAD expression and activity strongly correlate with poor PC clinical outcomes and increased DDR in biopsy RNA-seq studies. In contrast, miR-346 is associated with improved PC survival. INDUCE-seq reveals that miR-346-induced DSBs occur preferentially at binding sites of the most highly-transcriptionally active transcription factors in PC cells, including c-Myc, FOXA1, HOXB13, NKX3.1, and importantly, AR, resulting in target transcript downregulation. Further, RNA-seq reveals widespread miR-346 and shNORAD dysregulation of DNA damage, replication and cell cycle processes. NORAD drives target-directed miR decay (TDMD) of miR-346 as a novel genome protection mechanism: NORAD silencing increases mature miR-346 levels by several thousand-fold, and WT but not TDMD-mutant NORAD rescues miR-346-induced DNA damage. Importantly, miR-346 sensitises PC cells to DNA-damaging drugs including PARP inhibitor and chemotherapy, and induces tumour regression as a monotherapy in vivo, indicating that targeting miR-346:NORAD balance is a valid therapeutic strategy. CONCLUSIONS:A balancing act between miR-346 and NORAD regulates DNA damage and repair in PC. miR-346 may be particularly effective as a therapeutic in the context of decreased NORAD observed in advanced PC, and in transcriptionally-hyperactive cancer cells.

The concept of the histone code posits that histone modifications regulate gene functions once interpreted by epigenetic readers. A well-studied case is trimethylation of lysine 4 of histone H3 (H3K4me3), which is enriched at gene promoters. However, H3K4me3 marks are not needed for the expression of most genes, suggesting extra roles, such as influencing the 3D genome architecture. Here, we highlight an intriguing analogy between the H3K4me3-dependent induction of double-strand breaks in several recombination events and the impact of this same mark on DNA incisions for the repair of bulky lesions. We propose that Su(var)3-9, Enhancer-of-zeste and Trithorax (SET)-domain methyltransferases generate H3K4me3 to guide nucleases into chromatin spaces, the favorable accessibility of which ensures that DNA break intermediates are readily processed, thereby safeguarding genome stability.

Understanding how breaks form and are repaired in the genome depends on the accurate measurement of the frequency and position of DNA double strand breaks (DSBs). This is crucial for identification of a chemical's DNA damage potential and for safe development of therapies, including genome editing technologies. Current DSB sequencing methods suffer from high background levels, the inability to accurately measure low frequency endogenous breaks and high sequencing costs. Here we describe INDUCE-seq, which overcomes these problems, detecting simultaneously the presence of low-level endogenous DSBs caused by physiological processes, and higher-level recurrent breaks induced by restriction enzymes or CRISPR-Cas nucleases. INDUCE-seq exploits an innovative NGS flow cell enrichment method, permitting the digital detection of breaks. It can therefore be used to determine the mechanism of DSB repair and to facilitate safe development of therapeutic genome editing. We further discuss how the method can be adapted to detect other genomic features.

The interplay between active biological processes and DNA repair is central to mutagenesis. Here, we show that the ubiquitous process of replication initiation is mutagenic, leaving a specific mutational footprint at thousands of early and efficient replication origins. The observed mutational pattern is consistent with two distinct mechanisms, reflecting the two-step process of origin activation, triggering the formation of DNA breaks at the center of origins and local error-prone DNA synthesis in their immediate vicinity. We demonstrate that these replication initiation-dependent mutational processes exert an influence on phenotypic diversity in humans that is disproportionate to the origins' genomic size: By increasing mutational loads at gene promoters and splice junctions, the presence of an origin significantly influences both gene expression and mRNA isoform usage. Last, we show that mutagenesis at origins not only drives the evolution of origin sequences but also contributes to sculpting regulatory domains of the human genome.

Abstract NORAD (NOn-Coding RNA Activated by DNA Damage) is a highly-abundant, evolutionarily-conserved lncRNA. It maintains mitosis, DNA damage repair (DDR), and chromosomal integrity through PUM1/2 sequestration (PUM1/2 activity increases turnover of DDR factors), and through formation of a TOPO2-containing complex critical for genome integrity. We show that NORAD activity is regulated by microRNA-346 (miR-346), which disrupts NORAD:PUM2, interaction, leading to PUM2 destabilization and derepression of PUM1/2 DDR targets in prostate cancer (PCa) cells. RNA-seq reveals widespread miR-346 dysregulation of DNA damage, DNA replication and cell cycle transcripts. A novel method for high resolution, amplification-free genome-wide mapping of double strand DNA breaks (DSBs) (INDUCE-seq) reveals miR-346 induces DSBs specifically at transcription start sites characterized by phospho-PolII/CTCF/ZFX binding - a phenomenon not previously described for any microRNA. Mechanistically, DSBs result from miR-346 activation of transcription, R-loop formation and replicative catastrophe. This results in rapid dose-dependent induction of DNA damage, leading to checkpoint activation and cytosolic DNA accumulation, rescuable by NORAD. This cytosolic DNA activates cytokine-inducing cGAS-STING/RIG-1 innate immune pathways. Indeed, RNA-seq analysis reveals the top NORAD-enriched pathway as interferon signaling, while cytokine arrays reveal secretion of pro-Treg, MDSC and TAM factors by NORAD-overexpressing cells. NORAD inversely correlates with tumor immune response in gene expression data sets, and expression-based immune infiltration scoring predicts increased M2 macrophages, and reduced NK, CD8+ve, Th1 and cytotoxic T cells in NORAD-high vs NORAD-low PCa, indicating an ‘immune-cold' microenvironment. Excitingly, NORAD silencing results in several thousand-fold increase in mature miR-346 without affecting pri-miR levels, supporting NORAD's ability to drive target-directed microRNA decay (TDMD) of miR-346 as a critical novel genome protection mechanism. However, miR-346-induced DNA damage is in part NORAD-independent, since miR-346 induces DSBs within 1h, and in contrast to most miRs is predominantly chromatin-bound (NORAD is cytoplasmic). Critically, miR-346 sensitizes PCa cells to DNA-damaging chemotherapy and PARP inhibition. MiR-346 expression is associated with improved PCa survival, and reduced in high vs low Gleason grade PCa. Notably, NORAD strongly correlates with DDR signatures in early-stage, but not advanced metastatic PCa. Despite its DDR-promoting activity, and in contrast to miR-346, it is associated with worse survival across multiple patient cohorts. In conclusion, these data demonstrate that the NORAD:miR-346 interaction determines DNA damage response and innate immune pathway activity to regulate tumor immune response in PCa. Citation Format: Claire Fletcher, Lin Deng, Folake Orafidiya, Wei Yuan, Ines Figueiredo, Bora Gurel, Damien Leach, Fadi Issa, Antje Neeb, Denisa Bogdan, Felix Dobbs, Yiannis Philippou, Emma A. Murphy, Shuang G. Zhao, Joanna Hester, Richard J. Bryant, Simon H. Reed, Karen E. Knudsen, Ian G. Mills, Johann de Bono, Charlotte L. Bevan. Long non-coding RNA NORAD interaction with miR-346 impacts DNA damage response and anti-tumor immunity in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2362.

Background Local sequence context is known to have an impact on the mutational pattern seen in cancer. The RAS genes and a smoking carcinogen, Benzo[a]pyrene diol epoxide (BPDE), have been utilised to explore these context effects. BPDE is known to form an adduct at the guanines in a number of RAS gene sites, KRAS codons 12, 13 and 14, NRAS codon 12, and HRAS codons 12 and 14. Results Molecular modelling techniques, along with multivariate analysis, have been utilised to determine the sequence influenced differences between BPDE-adducted RAS gene sequences as well as the local distortion caused by the adducts. Conclusions We conclude that G:C > T:A mutations at KRAS codon 12 in the tumours of lung cancer patients (who smoke), proposed to be predominantly caused by BPDE, are due to the effect of the interaction methyl group at the C5 position of the thymine base in the KRAS sequence with the BPDE carcinogen investigated causing increased distortion. We further suggest methylated cytosine would have a similar effect, showing the importance of methylation in cancer development.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Despite recent advances in our understanding of the function of long noncoding RNAs (lncRNAs), their roles and functions in DNA repair pathways remain poorly understood. By screening a panel of uncharacterized lncRNAs to identify those whose transcription is induced by double-strand breaks (DSBs), we identified a novel lncRNA referred to as LRIK that interacts with Ku, which enhances the ability of the Ku heterodimer to detect the presence of DSBs. Here, we show that depletion of LRIK generates significantly enhanced sensitivity to DSB-inducing agents and reduced DSB repair efficiency. In response to DSBs, LRIK enhances the recruitment of repair factors at DSB sites and facilitates γH2AX signaling. Our results demonstrate that LRIK is necessary for efficient repairing DSBs via nonhomologous end-joining pathway.

The genetic information contained within the DNA molecule is highly susceptible to chemical and physical insult, caused by both endogenous and exogenous sources that can generate in the order of thousands of lesions a day in each of our cells (Lindahl, Nature 362(6422):709-715, 1993). DNA damages interfere with DNA metabolic processes such as transcription and replication and can be potent inhibitors of cell division and gene expression. To combat these regular threats to genome stability, a host of DNA repair mechanisms have evolved. When DNA lesions are left unrepaired due to defects in the repair pathway, mutations can arise that may alter the genetic information of the cell. DNA repair is thus fundamental to genome stability and defects in all the major repair pathways can lead to cancer predisposition. Therefore, the ability to accurately measure DNA damage at a genomic scale and determine the level, position, and rates of removal by DNA repair can contribute greatly to our understanding of how DNA repair in chromatin is organized throughout the genome. For this reason, we developed the 3D-DIP-Chip protocol described in this chapter. Conducting such measurements has potential applications in a variety of other fields, such as genotoxicity testing and cancer treatment using DNA damage inducing chemotherapy. Being able to detect and measure genomic DNA damage and repair patterns in individuals following treatment with chemotherapy could enable personalized medicine by predicting response to therapy.