Abstract Crossovers are essential for accurate chromosome segregation in meiosis. Yet the programmed DNA double-strand breaks that initiate them frequently occur in genes and pose a risk to transcription required for gametogenesis. How meiotic cells reconcile these competing demands has remained unclear. Here, we generate a genome-wide in vivo atlas of meiotic recombination intermediates across ∼42,000 hotspots by mapping repair proteins BLM, HFM1, and RPA in wild-type and genome-engineered mutant mouse testes. These maps reveal two distinct modes of break repair: a fast-resolving class with short-lived intermediates that are repaired predominantly as non-crossovers, and a slower class with persistent intermediates that give rise to nearly all crossovers. Fast-resolving hotspots occur almost exclusively within a deeply conserved set of ∼4,500 genes marked by structural and chromatin features established during an early stage of meiotic transcription. This transcriptional memory predicts repair fate with high accuracy across mouse subspecies and sexes. Across widely diverged mammals, including humans and cattle, orthologous genes show similar crossover suppression. Our findings reveal an early bifurcation between crossover and non-crossover repair that is governed by the transcriptional context of meiotic breaks. Together, they establish an evolutionarily conserved principle in which crossovers are directed away from transcriptionally important genes, thereby safeguarding gene function and shaping their evolution.

Cholangiocarcinoma (CCA) is a lethal cancer of the bile duct and is a major health concern in several parts of the world, including northeastern Thailand, where CCA incidence is the highest due to the endemic liver fluke Opisthorchis viverrini. Multiple studies have characterised genomic alterations in CCA tumours, and specific chromosomal alterations can predict prognosis. However, it is not known whether chromosomal instability (CIN), ongoing genomic alteration characteristic of most cancer types, is present in CCA tumours. In this study we leveraged a panel of cancer cell lines derived from fluke-positive CCA patients, as well as a matched normal cholangiocyte line as a control, to characterise CIN in CCA. We observed elevated rates of chromosome segregation errors compared to normal cells, although overall CIN rates were lower than those for highly genomically unstable cancers, such as colorectal or ovarian cancer. Chromosome segregation errors in CCA cell lines were potentially driven by elevated DNA replication stress and centrosome duplication. Single-cell genome sequencing and karyotyping of the cell lines showed extensive structural and numerical chromosomal aberrations, as well as copy number alterations that were heterogeneous between individual cells, supporting the presence of ongoing CIN in these cell line models. Low-pass whole-genome sequencing of 33 CCA tumour samples with matched normal tissue from northeastern Thailand, a liver fluke-endemic region showed increased whole and subchromosomal level alterations, with a higher extent of genomic alterations in intrahepatic tumours compared to extrahepatic. Eight tumours carried focal amplifications and/or deletions involving known cancer genes, as well as potential chromosomal instability-associated genes, including CCNE1 amplifications and a rare amplification of BRCA1. This study provides increased understanding of the rate and potential mechanisms of CIN in CCA that may inform new therapeutic strategies that synergise with specific ongoing CIN mechanisms. © 2025 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Horizontal transfer of nuclear DNA between cells of host and cancer is a potential source of adaptive variation in cancer cells. An understanding of the frequency and significance of this process in naturally occurring tumors is, however, lacking. We screened for this phenomenon in the transmissible cancers of dogs and Tasmanian devils and found an instance in the canine transmissible venereal tumor (CTVT). This involved introduction of a 15-megabase dicentric genetic element, composed of 11 fragments of six chromosomes, to a CTVT sublineage occurring in Asia around 2,000 y ago. The element forms the short arm of a small submetacentric chromosome and derives from a dog with ancestry associated with the ancient Middle East. The introduced DNA fragment is transcriptionally active and has adopted the expression profile of CTVT. Its features suggest that it may derive from an engulfed apoptotic body. Our findings indicate that nuclear horizontal gene transfer, although likely a rare event in tumor evolution, provides a viable mechanism for the acquisition of genetic material in naturally occurring cancer genomes.

Although somatic cell genomes are usually entirely clonally inherited, nuclear DNA exchange between cells of an organism can occur sporadically by cell fusion, phagocytosis or other mechanisms. This phenomenon has long been noted in the context of cancer, where it could be envisaged that DNA horizontal transfer plays a functional role in disease evolution. However, an understanding of the frequency and significance of this process in naturally occurring tumours is lacking. The host-tumour genetic discordance of transmissible cancers, malignant clones which pass between animals as allogeneic grafts, provides an opportunity to investigate this. We screened for host-to-tumour horizontal transfer of nuclear DNA in 174 tumours from three transmissible cancers affecting dogs and Tasmanian devils, and detected a single instance in the canine transmissible venereal tumour (CTVT). This involved introduction of a 15-megabase dicentric genetic element, composed of 11 rearranged fragments of six chromosomes, to a CTVT sublineage occurring in Asia around 2,000 years ago. The element forms the short arm of a small submetacentric chromosome, and derives from a dog with ancestry associated with the ancient Middle East. The introduced DNA fragment is transcriptionally active and has adopted the expression profile of CTVT. Its 143 genes do not, however, confer any obvious advantage to its spatially restricted CTVT sublineage. Our findings indicate that nuclear DNA horizontal transfer, although likely a rare event in tumour evolution, provides a viable mechanism for the acquisition of genetic material in naturally occurring cancer genomes. ### Competing Interest Statement The authors have declared no competing interest.

Gene therapy for xeroderma pigmentosum (XP), a rare, recessive DNA repair disease, has been considered since defects in XP genes result in severe and debilitating symptoms. Mutations in the XPC DNA repair gene result in a more that 1000-fold increased sensitivity to sunlight-induced skin cancer. The XPC gene is large (33 Kb) and the entire genomic locus is a difficult candidate for many gene therapy vectors to incorporate into their system by conventional cloning. Artificial chromosome vectors were developed to accommodate large genes and their regulatory sequences to allow full gene expression in cells. The HSV-1 human artificial chromosome (HAC) vectors we previously generated incorporated genes up to 100 Kb in a single vector. Subsequently, we modified the system to allow larger (>100 Kb) DNA gene sequences to be introduced by simultaneously infecting cells with two separate HSV-1 vector particles, one containing DNA required for HAC formation and the other with the desired gene. Following transduction, recombination of DNA formed a gene expressing HAC in vitro. The dual transduction system was successful for introduction and expression of the HPRT gene in human 3D engineered tissues and stem cells. In this study, we report the XPC gene delivery and transient gene expression via the dual transduction system in human cultured fibrosarcoma (HT1080) and primary XPC deficient patient cells.

Cell replacement therapies for ocular diseases characterised by photoreceptors degeneration are challenging due to poor primary cell survival in culture. A stable retinal cell source to replace lost photoreceptors holds promise. Müller glia cells play a pivotal role in retinal homoeostasis by providing metabolic and structural support to retinal neurons, preventing aberrant photoreceptors migration, and facilitating safe glutamate uptake. In fish and amphibians, injured retinas regenerate due to Müller-like glial stem cells, a phenomenon absent in the mammalian retina for unknown reasons. Research on Müller cells has been complex due to difficulties in obtaining pure cell population and their rapid de-differentiation in culture. While various Müller glia cell lines from human and rats are described, no nonhuman primate Müller glia cell line is currently available. Here, we report spontaneously immortalised Müller glia cell lines derived from macaque neural retinas that respond to growth factors and expand indefinitely in culture. They exhibit Müller cells morphology, such as an elongated shape and cytoplasmic projections, express Müller glia markers (VIMENTIN, GLUTAMINE SYNTHASE, glutamate-aspartate transporter, and CD44), and express stem cell markers such as PAX6 and SOX2. In the presence of factors that induce photoreceptor differentiation, these cells show a shift in gene expression patterns suggesting a state of de-differentiation, a phenomenon known in reprogrammed mammalian Müller cells. The concept of self-renewing retina might seem unfeasible, but not unprecedented. While vertebrate Müller glia have a regeneration potential absent in mammals, understanding the mechanisms behind reprogramming of Müller glia in mammals could unlock their potential for treating retinal degenerative diseases.

Understanding the genetic and nongenetic determinants of tumor protein 53 ( TP53 ) - mutation-driven clonal evolution and subsequent transformation is a crucial step toward the design of rational therapeutic strategies. Here we carry out allelic resolution single-cell multi-omic analysis of hematopoietic stem/progenitor cells (HSPCs) from patients with a myeloproliferative neoplasm who transform to TP53- mutant secondary acute myeloid leukemia (sAML). All patients showed dominant TP53 ‘multihit’ HSPC clones at transformation, with a leukemia stem cell transcriptional signature strongly predictive of adverse outcomes in independent cohorts, across both TP53- mutant and wild-type (WT) AML. Through analysis of serial samples, antecedent TP53 -heterozygous clones and in vivo perturbations, we demonstrate a hitherto unrecognized effect of chronic inflammation, which suppressed TP53 WT HSPCs while enhancing the fitness advantage of TP53- mutant cells and promoted genetic evolution. Our findings will facilitate the development of risk-stratification, early detection and treatment strategies for TP53 -mutant leukemia, and are of broad relevance to other cancer types.

One supplemental movie, four supplemental tables, six supplemental figures, supplemental methods

Mammalian meiotic recombination proceeds via repair of hundreds of programmed DNA double-strand breaks, which requires choreographed binding of RPA, DMC1, and RAD51 to single-stranded DNA substrates. High-resolution in vivo binding maps of these proteins provide insights into the underlying molecular mechanisms. When assayed in F 1 -hybrid mice, these maps can distinguish the broken chromosome from the chromosome used as template for repair, revealing more mechanistic detail and enabling the structure of the recombination intermediates to be inferred. By applying CRISPR-Cas9 mutagenesis directly on F 1 -hybrid embryos, we have extended this approach to explore the molecular detail of recombination when a key component is knocked out. As a proof of concept, we have generated hybrid biallelic knockouts of Dmc1 and built maps of meiotic binding of RAD51 and RPA in them. DMC1 is essential for meiotic recombination, and comparison of these maps with those from wild-type mice is informative about the structure and timing of critical recombination intermediates. We observe redistribution of RAD51 binding and complete abrogation of D-loop recombination intermediates at a molecular level in Dmc1 mutants. These data provide insight on the configuration of RPA in D-loop intermediates and suggest that stable strand exchange proceeds via multiple rounds of strand invasion with template switching in mouse. Our methodology provides a high-throughput approach for characterization of gene function in meiotic recombination at low animal cost.

Hereditary fibrosing poikiloderma (HFP) is a rare human dominant negative disorder caused by mutations in the FAM111B gene that encodes a nuclear trypsin-like serine protease. HFP patients present with symptoms including skin abnormalities, tendon contractures, myopathy and lung fibrosis. We characterized the cellular roles of human FAM111B using U2OS and MCF7 cell lines and report here that the protease interacts with components of the nuclear pore complex. Loss of FAM111B expression resulted in abnormal nuclear shape and reduced telomeric DNA content suggesting that FAM111B protease is required for normal telomere length; we show that this function is independent of telomerase or recombination driven telomere extension. Even though FAM111B-deficient cells were proficient in DNA repair, they showed hallmarks of genomic instability such as increased levels of micronuclei and ultra-fine DNA bridges. When mutated as in HFP, FAM111B was more frequently localized to the nuclear envelope, suggesting that accumulation of the mutated protease at the nuclear periphery may drive the disease pathology.

Sterility or subfertility of male hybrid offspring is commonly observed. This phenomenon contributes to reproductive barriers between the parental populations, an early step in the process of speciation. One frequent cause of such infertility is a failure of proper chromosome pairing during male meiosis. In subspecies of the house mouse, the likelihood of successful chromosome synapsis is improved by the binding of the histone methyltransferase PRDM9 to both chromosome homologs at matching positions. Using genetic manipulation, we altered PRDM9 binding to occur more often at matched sites, and find that chromosome pairing defects can be rescued, not only in an intersubspecific cross, but also between distinct species. Using different engineered variants, we demonstrate a quantitative link between the degree of matched homolog binding, chromosome synapsis, and rescue of fertility in hybrids between Mus musculus and Mus spretus. The resulting partial restoration of fertility reveals additional mechanisms at play that act to lock-in the reproductive isolation between these two species.

Abstract Background Exhaled breath analysis is a promising approach for oesophageal adenocarcinoma (OAC) early detection. The biomarkers of interest are low molecular weight metabolites including volatile aldehydes. In this translational study we investigated whether these metabolites originated from a tumoral source, and how this might impact the diagnosis and treatment of OAC patients. Methods The investigative strategy was directed by an unbiased informatics screen of metabolic reprogramming in OAC, and validated using complimentary gene expression assays (n = 638, including controls). Mass spectrometric methods were used to quantify corresponding metabolites and putative source compounds at a tissue level (n = 158), and also in exhaled breath for correlative purposes. Targeted in vitro experiments were performed to demonstrate the cause and effect of the proposed model of metabolic reprogramming in OAC. Results The unbiased screen and subsequent validation found that reduced aldehyde detoxification is an OAC hallmark. In vitro and in vivo this was associated with endogenous aldehyde accumulation. OAC tissue was generally enriched for volatile aldehydes, including the genotoxins formaldehyde, acetaldehyde, 4-hydroxy-2-nonenal and 2-butenal, and the exhaled biomarker decanal (all P < 0.0001). Decanal concentrations correlated with exhaled concentrations. Considering potential aldehyde sources, the OAC phospholipidome was characterised by desaturated and longer lipid acyls, and these spontaneously generated biomarker aldehyde species at ambient conditions. Enriched genotoxic aldehydes were detectable in base-pairing positions in DNA; this genotoxicity was therapeutically targetable with aldehyde scavengers in vitro. Conclusions These data support a model for enriched exhaled aldehydes based on increased production from an altered lipid phenotype, and reduced detoxification. Some aldehydes are non-reactive and thus support non-invasive detection. Others react with DNA and increase local genotoxicity; this process is druggable. These findings have implications for OAC early diagnosis and chemoprevention.

For the production and rederivation of mouse strains, pseudopregnant female mice are used for embryo transfer and serve as surrogate mothers to support embryo development to term. Vasectomized males are commonly used to render pseudopregnancy in females, generated by surgical procedures associated with considerable pain and discomfort. Genetically modified mouse strains with a sterility phenotype provide a non-surgical replacement and represent an important application of the 3Rs (Replacement, Reduction, Refinement). However, the maintenance of such genetically modified mouse strains requires extensive breeding and genotyping procedures, which are regulated procedures under national legislation. As an alternative, we have explored the use of sterile male hybrids that result when two wild-type mouse subspecies, Mus musculus musculus and Mus musculus domesticus , interbreed. We find the male STUSB6F1 hybrid, resulting from the mating of female STUS/Fore with male C57BL/6J, ideally suited and demonstrate that its performance for the production of oviduct and uterine transfer recipients is indistinguishable when compared to surgically vasectomized mice. The use of these sterile hybrids avoids the necessity for surgical procedures or the breeding of sterile genetically modified lines and can be generated by the simple mating of two wild-type laboratory strains—a non-regulated procedure. Furthermore, in contrast with the breeding of genetically sterile mice, all male offspring are sterile and suitable for the generation of pseudopregnancy, allowing their efficient production with minimal breeding pairs.

Chromosome instability (CIN) consists of high rates of structural and numerical chromosome abnormalities and is a well-known hallmark of cancer. Aluminum is added to many industrial products of frequent use. Yet, it has no known physiological role and is a suspected human carcinogen. Here, we show that V79 cells, a well-established model for the evaluation of candidate chemical carcinogens in regulatory toxicology, when cultured in presence of aluminum-in the form of aluminum chloride (AlCl3) and at concentrations in the range of those measured in human tissues-incorporate the metal in a dose-dependent manner, predominantly accumulating it in the perinuclear region. Intracellular aluminum accumulation rapidly leads to a dose-dependent increase in DNA double strand breaks (DSB), in chromosome numerical abnormalities (aneuploidy) and to proliferation arrest in the G2/M phase of the cell cycle. During mitosis, V79 cells exposed to aluminum assemble abnormal multipolar mitotic spindles and appear to cluster supernumerary centrosomes, possibly explaining why they accumulate chromosome segregation errors and damage. We postulate that chronic aluminum absorption favors CIN in mammalian cells, thus promoting carcinogenesis.

Volatile aldehydes are enriched in esophageal adenocarcinoma (EAC) patients’ breath and could improve early diagnosis, however the mechanisms of their production are unknown. Here, we show that weak aldehyde detoxification characterizes EAC, which is sufficient to cause endogenous aldehyde accumulation in vitro. Two aldehyde groups are significantly enriched in EAC biopsies and adjacent tissue: (i) short-chain alkanals, and (ii) medium-chain alkanals, including decanal. The short-chain alkanals form DNA-adducts, which demonstrates genotoxicity and confirms inadequate detoxification. Metformin, a putative aldehyde scavenger, reduces this toxicity. Tissue and breath concentrations of the medium-chain alkanal decanal are correlated, and increased decanal is linked to reduced ALDH3A2 expression, TP53 deletion, and adverse clinical features. Thus, we present a model for increased exhaled aldehydes based on endogenous accumulation from reduced detoxification, which also causes therapeutically actionable genotoxicity. These results support EAC early diagnosis trials using exhaled aldehyde analysis.

Abstract Background Genome editing in mice using either classical approaches like homologous recombination or CRISPR/Cas9 has been reported to harbor off target effects (insertion/deletion, frame shifts or gene segment duplications) that lead to mutations not only in close proximity to the target site but also outside. Only the genomes of few engineered mouse strains have been sequenced. Since the role of the ether-lipid cleaving enzyme alkylglycerol monooxygenase (AGMO) in physiology and pathophysiology remains enigmatic, we created a knockout mouse model for AGMO using EUCOMM stem cells but unforeseen genotyping issues that did not agree with Mendelian distribution and enzyme activity data prompted an in-depth genomic validation of the mouse model. Results We report a gene segment tandem duplication event that occurred during the generation of an Agmo knockout-first allele by homologous recombination. Only low homology was seen between the breakpoints. While a single copy of the recombinant 18 kb cassette was integrated correctly around exon 2 of the Agmo gene, whole genome nanopore sequencing revealed a 94 kb duplication in the Agmo locus that contains Agmo wild-type exons 1–3. The duplication fooled genotyping by routine PCR, but could be resolved using qPCR-based genotyping, targeted locus amplification sequencing and nanopore sequencing. Despite this event, this Agmo knockout mouse model lacks AGMO enzyme activity and can therefore be used to study its physiological role. Conclusions A duplication event occurred at the exact locus of the homologous recombination and was not detected by conventional quality control filters such as FISH or long-range PCR over the recombination sites. Nanopore sequencing provides a cost convenient method to detect such underrated off-target effects, suggesting its use for additional quality assessment of gene editing in mice and also other model organisms.

High-grade serous ovarian carcinoma is characterised by TP53 mutation and extensive chromosome instability (CIN). Because our understanding of CIN mechanisms is based largely on analysing established cell lines, we developed a workflow for generating ex vivo cultures from patient biopsies to provide models that support interrogation of CIN mechanisms in cells not extensively cultured in vitro. Here, we describe a "living biobank" of ovarian cancer models with extensive replicative capacity, derived from both ascites and solid biopsies. Fifteen models are characterised by p53 profiling, exome sequencing and transcriptomics, and karyotyped using single-cell whole-genome sequencing. Time-lapse microscopy reveals catastrophic and highly heterogeneous mitoses, suggesting that analysis of established cell lines probably underestimates mitotic dysfunction in advanced human cancers. Drug profiling reveals cisplatin sensitivities consistent with patient responses, demonstrating that this workflow has potential to generate personalized avatars with advantages over current pre-clinical models and the potential to guide clinical decision making. High-grade serous ovarian carcinoma is often associated with TP53 mutation and chromosomal instability (CIN). Here, the authors generate ex vivo cultures from biopsies and ascites of patients and perform characterization to evaluate CIN mechanisms and compare drug sensitivity with patient responses.

Gene expression studies and gene therapy require efficient gene delivery into cells. Different technologies by viral and non-viral mechanisms have been used for gene delivery into cells. Small gene vectors transfer across the cell membrane with a relatively high efficiency, but not large genes or entire loci spanning several kilobases, which do not remain intact following introduction. Previously, we developed an efficient delivery system based on herpes virus simplex type 1 (HSV-1) amplicons to transfer large fragments of DNA incorporated in human artificial chromosome (HAC) vectors into the nucleus of human cells. The HSV-1 amplicon lacks the signals for cleavage and replication of its own genome, yet each amplicon has the capacity to incorporate up to 150 kb of exogenous DNA. In this study, we investigated whether the capacity of gene delivery could be increased by simultaneously introducing multiple HSV-1 modified amplicons carrying a gene expressing HAC vector into cells with the aim of generating a single artificial chromosome containing the desired genes. Following co-transduction of two HSV-1 HAC amplicons, artificial chromosomes were successfully generated containing the introduced genes, which were appropriately expressed in different human cell types.