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All organisms possess molecular mechanisms that govern DNA repair and associated DNA damage response (DDR) processes. Owing to their relevance to human disease, most notably cancer, these mechanisms have been studied extensively, yet new DNA repair and/or DDR factors and functional interactions between them are still being uncovered. The emergence of CRISPR technologies and CRISPR-based genetic screens has enabled genome-scale analyses of gene–gene and gene–drug interactions, thereby providing new insights into cellular processes in distinct DDR-deficiency genetic backgrounds and conditions. In this Review, we discuss the mechanistic basis of CRISPR–Cas genetic screening approaches and describe how they have contributed to our understanding of DNA repair and DDR pathways. We discuss how DNA repair pathways are regulated, and identify and characterize crosstalk between them. We also highlight the impacts of CRISPR-based studies in identifying novel strategies for cancer therapy, and in understanding, overcoming and even exploiting cancer-drug resistance, for example in the contexts of PARP inhibition, homologous recombination deficiencies and/or replication stress. Lastly, we present the DDR CRISPR screen (DDRcs) portal, in which we have collected and reanalysed data from CRISPR screen studies and provide a tool for systematically exploring them.CRISPR-based genetic screens are providing new insights into the consequences of deficiencies in DNA damage response and repair pathways. These include insights into the regulation of homologous recombination and of replication stress and their crosstalk with other repair pathways, into novel cancer therapies and into the basis of cancer-drug resistance.

Key Points Chromothripsis is a phenomenon by which tens to thousands of chromosomal rearrangements occur, with the available evidence indicating that chromothripsis can be generated by a single catastrophic event during the life history of a cell. Rearrangements can occur by chromosome shattering and rejoining of pieces by end-joining DNA repair pathways, or by aberrant DNA replication-based mechanisms. Chromothripsis may contribute to cellular transformation, as it occurs early in tumour development: end-joining-based repair can lead to the loss of tumour suppressor functions, oncogenic fusions and oncogene amplification via double-minute chromosomes. In addition, aberrant DNA replication mechanisms taking place during chromothripsis can lead to oncogene amplification. An attractive model for the generation of chromothripsis invokes the involvement of micronuclei. According to this model, chromosomes contained within micronuclei suffer aberrant DNA replication and can then be pulverized in mitosis with subsequent rejoining of DNA segments leading to a derivative chromosome or chromosomes that can be reincorporated into the main nucleus. Chromothripsis is observed with a higher frequency in cells with mutated p53. This leads to a model in which micronuclei formation owing to chromosome segregation errors is allowed in p53-deficient cells, potentially yielding chromothripsis and the evolution of cancer. Defects in chromosome segregation and/or DNA damage response processes may also contribute to carcinogenesis by promoting chromothripsis. Chromothripsis is an emerging phenomenon that results in chromosome rearrangements in tumour cells. This Review discusses the possible mechanisms underlying this process and its implications for cancer biology and in the clinic. Genomic alterations that lead to oncogene activation and tumour suppressor loss are important driving forces for cancer development. Although these changes can accumulate progressively during cancer evolution, recent studies have revealed that many cancer cells harbour chromosomes bearing tens to hundreds of clustered genome rearrangements. In this Review, we describe how this striking phenomenon, termed chromothripsis, is likely to arise through chromosome breakage and inaccurate reassembly. We also discuss the potential diagnostic, prognostic and therapeutic implications of chromothripsis in cancer.

Down-regulation and mutations of the nuclear-architecture proteins lamin A and C cause misshapen nuclei and altered chromatin organization associated with cancer and laminopathies, including the premature-aging disease Hutchinson-Gilford progeria syndrome (HGPS). Here, we identified the small molecule \"Remodelin\" that improved nuclear architecture, chromatin organization, and fitness of both human lamin A/C–depleted cells and HGPS-derived patient cells and decreased markers of DNA damage in these cells. Using a combination of chemical, cellular, and genetic approaches, we identified the acetyl-transferase protein NAT10 as the target of Remodelin that mediated nuclear shape rescue in laminopathic cells via microtubule reorganization. These findings provide insights into how NAT10 affects nuclear architecture and suggest alternative strategies for treating laminopathies and aging.

The DNA mutation produced by cellular repair of a CRISPR-Cas9-generated double-strand break determines its phenotypic effect. It is known that the mutational outcomes are not random, but depend on DNA sequence at the targeted location. Here we systematically study the influence of flanking DNA sequence on repair outcome by measuring the edits generated by >40,000 guide RNAs (gRNAs) in synthetic constructs. We performed the experiments in a range of genetic backgrounds and using alternative CRISPR-Cas9 reagents. In total, we gathered data for >109 mutational outcomes. The majority of reproducible mutations are insertions of a single base, short deletions or longer microhomology-mediated deletions. Each gRNA has an individual cell-line-dependent bias toward particular outcomes. We uncover sequence determinants of the mutations produced and use these to derive a predictor of Cas9 editing outcomes. Improved understanding of sequence repair will allow better design of gene editing experiments.

DNA double-strand breaks (DSBs) may be repaired either by homologous recombination (HR) or nonhomologous end joining (NHEJ) pathways. A new high-resolution mapping study of DSBs in human cells shows that trimethylated histone H3 K36, a marker of active chromatin, targets RAD51 to DSBs within transcribed regions to promote preferential HR-mediated repair at transcriptionally active loci. Although both homologous recombination (HR) and nonhomologous end joining can repair DNA double-strand breaks (DSBs), the mechanisms by which one of these pathways is chosen over the other remain unclear. Here we show that transcriptionally active chromatin is preferentially repaired by HR. Using chromatin immunoprecipitation–sequencing (ChIP-seq) to analyze repair of multiple DSBs induced throughout the human genome, 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 are targeted to HR repair via the transcription elongation–associated mark trimethylated histone H3 K36. Concordantly, depletion of SETD2, the main H3 K36 trimethyltransferase, severely impedes HR at such DSBs. Our study thereby demonstrates a primary role in DSB repair of the chromatin context in which a break occurs.

Repair of single-ended DNA double-strand breaks (seDSBs) by homologous recombination (HR) requires the generation of a 3′ single-strand DNA overhang by exonuclease activities in a process called DNA resection. However, it is anticipated that the highly abundant DNA end-binding protein Ku sequesters seDSBs and shields them from exonuclease activities. Despite pioneering works in yeast, it is unclear how mammalian cells counteract Ku at seDSBs to allow HR to proceed. Here we show that in human cells, ATM-dependent phosphorylation of CtIP and the epistatic and coordinated actions of MRE11 and CtIP nuclease activities are required to limit the stable loading of Ku on seDSBs. We also provide evidence for a hitherto unsuspected additional mechanism that contributes to prevent Ku accumulation at seDSBs, acting downstream of MRE11 endonuclease activity and in parallel with MRE11 exonuclease activity. Finally, we show that Ku persistence at seDSBs compromises Rad51 focus assembly but not DNA resection. Homologous recombination requires end resection of the DNA at the site of the break, however the Ku dimer can sequester single-ended double-strand breaks. Here the authors show that ATM-dependent phosphorylation of CtIP, along with the actions of Mre11, impair the stable loading of Ku onto DNA.

The diversity of somatic mutations in human cancers can be decomposed into individual mutational signatures, patterns of mutagenesis that arise because of DNA damage and DNA repair processes that have occurred in cells as they evolved towards malignancy. Correlations between mutational signatures and environmental exposures, enzymatic activities and genetic defects have been described, but human cancers are not ideal experimental systems—the exposures to different mutational processes in a patient’s lifetime are uncontrolled and any relationships observed can only be described as an association. Here, we demonstrate the proof-of-principle that it is possible to recreate cancer mutational signatures in vitro using CRISPR-Cas9-based gene-editing experiments in an isogenic human-cell system. We provide experimental and algorithmic methods to discover mutational signatures generated under highly experimentally-controlled conditions. Our in vitro findings strikingly recapitulate in vivo observations of cancer data, fundamentally validating the concept of (particularly) endogenously-arising mutational signatures. As cells evolve towards malignancy, somatic mutations arise from defects in DNA damage and repair processes which are each associated with individual mutation signatures. Here the authors show it is possible to recreate cancer mutational signatures in vitro using gene editing experiments in an isogenic human-cell system.

Inhibiting DNA repair can have a positive outcome on therapeutic interventions All the cells in our bodies suffer many thousands of DNA lesions every day ( 1 ). The vast majority of these lesions are safely dealt with by cellular DNA repair and associated DNA damage response (DDR) activities that are, as a consequence, vital for life. Defects in or deregulation of our DNA repair/DDR systems are linked to many human pathologies ( 2 ). Yet, perhaps counterintuitively, pharmacological inhibitors of DNA repair/DDR have considerable potential in treating various human diseases, particularly cancer.

How changes in chromatin can modulate the repair pathway of DNA double-strand breaks is now investigated. The work shows that histone deacetylases HDAC1 and HDAC2 are recruited to sites of DNA damage, where they mediate the removal of H3K56 acetyl marks, and their activity is important for repair via non-homologous end-joining. DNA double-strand break (DSB) repair occurs within chromatin and can be modulated by chromatin-modifying enzymes. Here we identify the related human histone deacetylases HDAC1 and HDAC2 as two participants in the DNA-damage response. We show that acetylation of histone H3 Lys56 (H3K56) was regulated by HDAC1 and HDAC2 and that HDAC1 and HDAC2 were rapidly recruited to DNA-damage sites to promote hypoacetylation of H3K56. Furthermore, HDAC1- and 2-depleted cells were hypersensitive to DNA-damaging agents and showed sustained DNA-damage signaling, phenotypes that reflect defective DSB repair, particularly by nonhomologous end-joining (NHEJ). Collectively, these results show that HDAC1 and HDAC2 function in the DNA-damage response by promoting DSB repair and thus provide important insights into the radio-sensitizing effects of HDAC inhibitors that are being developed as cancer therapies.

SUMO modification and the response to DNA damage The occurrence of a double-strand break in DNA activates a complex series of events that recruit to the break many proteins involved in its repair. A number of these proteins are modified by addition of a small protein, SUMO; this modification is performed SUMO ligases. In this work, Jackson and colleagues show that two such ligases, PIAS1 and PIAS4, add various SUMOs onto DNA repair proteins at double-strand breaks. The PIAS ligases are recruited via their SAP domains, and their activity is required for effective repair. SUMOylation by PIAS1 and PIAS4 is also necessary for the further modification of certain repair factors by ubiquitin, a somewhat larger protein adduct related to SUMO. The successive SUMOylation and ubiquitylation of repair proteins regulates their targeting to, and repair of, DNA breaks. Following the formation of a DNA double-strand break (DSB), cells activate the DNA-damage response and recruit a number of proteins to the lesion. Some of these proteins are modified by the attachment of small ubiquitin-related modifier (SUMO). Here, SUMO1, SUMO2 and SUMO3 are shown to accumulate at DSB sites in mammalian cells. SUMO1 and SUMO2/3 accrual requires the E3 ligase enzymes PIAS4 and PIAS1, which promote DSB repair. DNA double-strand breaks (DSBs) are highly cytotoxic lesions that are generated by ionizing radiation and various DNA-damaging chemicals. Following DSB formation, cells activate the DNA-damage response (DDR) protein kinases ATM, ATR and DNA-PK (also known as PRKDC). These then trigger histone H2AX (also known as H2AFX) phosphorylation and the accumulation of proteins such as MDC1, 53BP1 (also known as TP53BP1), BRCA1, CtIP (also known as RBBP8), RNF8 and RNF168/RIDDLIN into ionizing radiation-induced foci (IRIF) that amplify DSB signalling and promote DSB repair 1 , 2 . Attachment of small ubiquitin-related modifier (SUMO) to target proteins controls diverse cellular functions 3 , 4 , 5 , 6 . Here, we show that SUMO1, SUMO2 and SUMO3 accumulate at DSB sites in mammalian cells, with SUMO1 and SUMO2/3 accrual requiring the E3 ligase enzymes PIAS4 and PIAS1. We also establish that PIAS1 and PIAS4 are recruited to damage sites via mechanisms requiring their SAP domains, and are needed for the productive association of 53BP1, BRCA1 and RNF168 with such regions. Furthermore, we show that PIAS1 and PIAS4 promote DSB repair and confer ionizing radiation resistance. Finally, we establish that PIAS1 and PIAS4 are required for effective ubiquitin-adduct formation mediated by RNF8, RNF168 and BRCA1 at sites of DNA damage 7 , 8 , 9 , 10 , 11 . These findings thus identify PIAS1 and PIAS4 as components of the DDR and reveal how protein recruitment to DSB sites is controlled by coordinated SUMOylation and ubiquitylation.