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At least 19 recordsLinked to original sources

PubMed2026

CRISPR/Cas9 screening revealed BIRC6-AS1/BIRC6 mediates abiraterone resistance via NHEJ pathway-dependent A20 degradation in prostate cancer.

Abiraterone acetate is a standard-of-care therapy for prostate cancer (PCa). However, resistance frequently emerges, often characterized by the progression to AR-independent phenotypes. Employing a genome-wide CRISPR/Cas9 library screening strategy, we identified 523 long non-coding RNAs (lncRNAs) and 2,183 protein-coding genes as potential candidates associated with abiraterone resistance. Notably, a pair of sense-antisense genes, BIRC6-AS1/BIRC6, was identified as a significant contributor to abiraterone resistance, serving as a critical survival factor in AR-independent contexts. BIRC6-AS1 depletion led to a reduction in both the mRNA and protein levels of BIRC6. Moreover, depletion of either BIRC6-AS1 or BIRC6 enhanced the sensitivity of PCa cells to abiraterone in both in vitro and in vivo settings. Further investigation revealed that BIRC6-AS1 stabilized the mRNA of BIRC6 through interaction with ILF2. Suppression of either BIRC6-AS1 or BIRC6 attenuated non-homologous end joining (NHEJ) repair activity, resulting in the disassembly of 53BP1 foci at DNA damage sites and an increased accumulation of DNA damage, thereby exposing a vulnerability in AR-independent resistant cells. Mechanistically, BIRC6 interacted with A20 and facilitated the K48-linked ubiquitination and subsequent degradation of A20 at the K337 residue. Additionally, A20 knockdown effectively reversed the abiraterone sensitivity induced by BIRC6-AS1 depletion. Collectively, our study provides a landscape of lncRNAs and protein-coding genes associated with abiraterone resistance and suggests that targeting the BIRC6-AS1/BIRC6 axis represents a potential therapeutic strategy to eradicate AR-independent resistant tumors in prostate cancer.

Journal Article↗

PubMed2026

Loss of Ku70 promotes mononucleate conidiation and homologous recombination in Phanerochaete chrysosporium.

Lignin is a major constituent of lignocellulose and the most abundant aromatic biopolymer on earth. It provides plants with rigidity and protection, but its recalcitrant nature also presents a significant barrier to lignocellulose valorization. The white-rot fungus Phanerochaete chrysosporium is among nature's most efficient lignin degraders, and its ligninolytic capabilities have been subjected to intensive investigations. Genome editing with precision is crucial for elucidating the in vivo mechanisms of its ligninolytic actions, but genetic manipulations of P. chrysosporium are often plagued by imprecision. This technical nuisance is driven primarily by canonical non-homologous end joining (c-NHEJ), a DNA repair system that requires little homology and depends on the binding of the Ku70/Ku80 heterodimer to double-strand break (DSB) ends. Loss of Ku70 or Ku80 abolishes c-NHEJ and significantly improves genome editing precision in many filamentous fungi, but it has yet to be examined and exploited in P. chrysosporium. Here, we constructed a homozygous ku70Δ mutant in a meiotic homokaryon of clear genetic background. Loss of Ku70 minimally impacts growth but significantly increases homologous recombination frequency from ~2% to ~66%, with ~32% of the latter being homozygous. Unexpectedly, loss of Ku70 also promotes mononucleate conidiation, which may facilitate isolation of homozygous mutants. Taken together, our work provides a valuable genetic tool to understand and exploit P. chrysosporium's remarkable ligninolytic capabilities.IMPORTANCEGenome editing with precision is essential to unraveling the intricacies of P. chrysosporium's exceptional ligninolytic capabilities, but the available tools are generally imprecise due to the dominance of non-homologous recombination, a problem that is further exacerbated by the discontinuation of Novozyme 234. We tackle these challenges by reestablishing protoplast-based transformation with Lywallzyme as an alternative. Importantly, we demonstrate that inactivation of c-NHEJ by deleting ku70 significantly increases gene knockout efficiency and report the unexpected involvement of c-NHEJ in regulating the number of nuclei during conidiation. Our work paves the way for future ventures into understanding ligninolysis in P. chrysosporium and building superior chassis for industrial applications.

Ku70↗

PubMed2025

γ-tubulin mediates DNA double-strand break repair.

Double-strand breaks (DSBs) in DNA pose a critical threat to genomic integrity, potentially leading to the onset and progression of various diseases, including cancer. Cellular responses to such lesions entail sophisticated repair mechanisms primarily mediated by non-homologous end joining (NHEJ) and homologous recombination (HR). Interestingly, the efficient recruitment of repair proteins and completion of DSB repair likely involve complex, inter-organelle communication and coordination of cellular components. In this study, we report a role of γ-tubulin in DSB repair. γ-tubulin is a major microtubule nucleation factor governing microtubule dynamics. We show that γ-tubulin is recruited to the site of DNA damage and is required for efficient DSB repair via both NHEJ and HR. Suppression of γ-tubulin impedes DNA repair and exacerbates DNA damage accumulation. Furthermore, γ-tubulin mediates the mobilization and formation of DNA damage foci, which serve as repair centers, thereby facilitating the recruitment of HR and NHEJ repair proteins on damaged chromatin. Finally, pharmacological inhibition of γ-tubulin enhances the cytotoxic effect of DNA-damaging agents, consistent with the DNA repair function of γ-tubulin, and underscoring the potential of its therapeutic intervention in cancer therapy.

DNA Breaks, Double-Stranded↗

PubMed2026

Characterization of a Ku-binding motif in the C-terminal region of RAG2.

We applied an unsupervised interactome analysis with the RAG2 C-terminal region (R2CT) in v-abl pro-B cells undergoing V(D)J recombination. Mass-spectrometry analyses showed that Ku70 and Ku80 were among the top 10 hits. To further strengthen these observations, we performed Proximity Ligation Assay (PLA) and characterize the existence of a GFP-R2CT-Ku complex formation in cellulo. The interaction of several partners with Ku70/80 (Ku) through Ku-binding motifs (KBMs) in their sequences governs their enrolment in NHEJ repair complexes. Through sequence analysis, we identified a KBM within R2CT (R-KBM, amino acids 589-527). We confirmed by calorimetry a specific micromolar interaction between this RAG2 region and Ku70/80/DNA complex. The RAG2 motif KBM can be subdivided in two conserved parts that have no interaction individually. AlphaFold2 prediction coupled with molecular dynamic simulations indicate that the C-terminal part of the RAG2 motif interacts with Ku80 on the same site than the NHEJ factor XLF. These in silico analyses indicated that the N-terminal part of the RAG2 motif interacts with DNA adjacent to Ku with a major role of the K503 residue in agreement with disruption of the interaction observed with the K503E mutant. This study further extends the large ensemble of proteins recruited at DSBs by KBM motifs and substantiates the model of a tight coupling between DNA breakage and repair during V(D)J recombination, mediated by the Ku-RAG2 C-terminus interaction.

Ku Autoantigen↗

PubMed2026

Non-syndromic premature ovarian insufficiency associated with monoallelic LIG4 mutation via haploinsufficiency.

BACKGROUND: Premature ovarian insufficiency (POI) is a heterogeneous reproductive disorder, with genetic factors, particularly defects in DNA damage response pathways, increasingly implicated in its pathogenesis. DNA ligase IV (LIG4) is a key enzyme in the non-homologous end joining (NHEJ) pathway responsible for repairing DNA double-strand breaks (DSBs). However, its role in non-syndromic POI remains unclear. This study aimed to investigate the potential contribution of LIG4 variants to non-syndromic POI. RESULTS: Whole-exome sequencing identified a heterozygous frameshift variant in LIG4 (c.1271_1275del) in a three-generation Han Chinese family with non-syndromic POI, which co-segregated with affected individuals. AlphaFold-based structural modeling predicted truncation of the C-terminal XRCC4 interaction region. Functional experiments demonstrated that the mutant LIG4 protein showed reduced stability and was predominantly mislocalized to the cytoplasm of cells. In ovarian KGN cells, LIG4 depletion reduced cell viability, induced stress-associated cellular senescence, and impaired DNA damage repair capacity. In LIG4 knockout 293T cells, co-transfection of wild-type and mutant constructs revealed dose-dependent functional impairment, resulting in increased apoptosis under basal conditions and after phleomycin induced DNA damage, together with delayed repair of DSBs. Reanalysis of public single-cell RNA sequencing data further showed stage specific upregulation of LIG4 during oocyte maturation. Co-expression network analysis revealed enrichment in the Fanconi anemia pathway, phosphatidylinositol 3-kinase signaling pathway, and glycan metabolism. CONCLUSIONS: Our findings suggest that monoallelic LIG4 mutations may represent a potential genetic etiology for non-syndromic POI with sex-limited penetrance. While further validation in more physiologically relevant models is warranted, our data indicate that LIG4 haploinsufficiency may impair DSB repair and disrupt molecular pathways crucial for oocyte maturation and survival, highlighting a potential role of the NHEJ pathway in maintaining human ovarian function.

Humans↗

PubMed2025

Efficiency of DNA repair mechanisms of domestic dog primary fibroblasts isolated from small and large breeds of different ages in response to double stranded breaks (DSB).

Aging is associated with increased genomic instability, a phenomenon largely driven by the accumulation of DNA damage over time, and large species of mammals seem to have more robust DNA repair systems associated with longer lives. Among DNA lesions, double-strand breaks (DSBs) are particularly deleterious and have been implicated in age-related functional decline and disease. In this study, we investigated how age and body mass affect the efficiency of DSB repair (DSBr) in primary fibroblast cells isolated from domestic dogs, a species that exhibits significant intraspecies variation in lifespan and body mass. Primary fibroblast cells were isolated from puppies and senior dogs of both large and small breeds. Cells were treated with 100 µM etoposide to induce DSBs and subsequently analyzed at two post-treatment recovery intervals (2 and 24 h) to correlated with the two pathways associated with DSBr, the fast, non-homologous end joining (NHEJ) and the much slower, homologous recombination (HR). Cells were stained for γ-H2AX foci and images were collected using confocal microscopy. We found that mean fluorescence per cell was higher in older dogs of both size classes in the 2 h recovery, indicating higher amounts of DNA damage but suggesting similar efficiencies through the NHEJ repair mechanism in older dogs despite size class. We also show that mean fluorescence per cell was higher in the older large breed dogs in the 24 h recovery, suggesting that the slower phase associated with HR seems to be deficient in cells from older, larger breeds of dogs. These findings support the broader theory that aging is associated with impaired genomic maintenance and establish domestic dogs as a valuable model for studying the cellular mechanisms of age-related genomic instability.

Animals↗

PubMed2026

Repair of DNA double-strand breaks after low radiation doses in childhood cancer survivors and matched cancer-free individuals.

DNA double-strand breaks (DSBs) which arise in G1- or G0-phase normal human cells are repaired by nonhomologous end-joining (NHEJ), a pathway which is important for cell survival but can cause mutations at the break sites. DSB repair by NHEJ is very efficient at high damage levels of 1 or more DSBs per cell, much less efficient at lower damage levels and almost absent if only ~0.05 DSBs per cell are induced. Here, we have analyzed the repair of high and low levels of radiation-induced DSBs in primary fibroblasts from 136 childhood cancer survivors, half of whom developed a second independent tumor later in life, and compared it to the response of primary fibroblasts from 68 individually matched cancer-free individuals. We measured the DSB repair efficiency by quantifying residual γH2AX foci with an automated scoring system at 24 h after irradiation with doses of 2.5, 5, 10, and 100 mGy, which induce about 0.0625, 0.125, 0.25, and 2.5 DSBs per cell, respectively. Although childhood cancer survivors and cancer-free individuals repaired DSBs after 10 and 100 mGy equally efficiently, their response to lower doses differed drastically. While repair in cancer-free individuals was inefficient after 2.5 mGy, childhood cancer survivors repaired DSBs after this dose as efficiently as after higher doses. These results indicate that most of the childhood cancer survivors analyzed here may harbor a genetic alteration that affects their response to low levels of DSBs. We suggest that such alterations may be either inherited or caused by previous tumor treatments.

Humans↗

PubMed2025

Engineering recombination machinery facilitates the construction of yeast cell factories.

Advances in genome editing have been promoted by programmable nucleases like CRISPR-Cas9, which triggers endogenous DNA repair mechanisms by inducing double-strand break (DSB). Cellular responses to DSBs are governed by competing repair pathways: error-prone non-homologous end joining (NHEJ) and high-fidelity homologous recombination (HR). This review systematically compares the molecular mechanisms and key regulators of NHEJ and HR, with a focus on recent breakthroughs in recombination engineering in non-conventional yeasts. These advances address challenges in precise genome editing, enabling robust metabolic engineering of yeast cell factories for sustainable bioproduction.

Metabolic Engineering↗

PubMed2025

Disruption of Microhomology-mediated End-joining in Ewing Sarcoma.

Ewing sarcoma (EwS) is a group of bone and soft tissue cancers in children and young adults. Since EwS cells have pronounced sensitivity to radiation and chemotherapy-induced DNA damage, the role of the oncoprotein, EWS-FLI1, in DNA repair is likely. Here, we demonstrate that EWS-FLI1 causes a defect in microhomology-mediated end-joining (MMEJ) repair. EWSR1 is a splicing factor that promotes the faithful splicing of the POLQ pre-mRNA, required for the expression of POLΘ, a critical protein in the MMEJ pathway. Expression of EWS-FLI1, or loss of EWSR1, causes exon 25 skipping of the POLQ transcript, decreased POLΘ expression, impaired MMEJ, and cellular sensitivity to inhibitors of the Fanconi Anemia (FA), NHEJ, or HR pathways, through the mechanism of synthetic lethality. Knockdown of EWS-FLI1 expression restores POLΘ mitotic foci and increases MMEJ activity. Inhibitors of the FA, NHEJ, or HR therefore may provide a targeted therapy for patients with EwS.

Alternative end-joining↗

PubMed2026

Whole genome and exome sequencing of pancreatic neuroendocrine tumour to investigate PRRT response.

Patients with pancreatic neuroendocrine tumours (PNETs) often have similar baseline clinical characteristics, including grade and molecular imaging phenotype, yet have highly variable responses to peptide receptor radionuclide therapy (PRRT). To identify genomic alterations and mutational patterns associated with PRRT treatment response and acquired somatic changes following PRRT exposure, whole genome or exome sequencing was applied to 40 PNET samples from 32 patients, including eight paired pre- or post-PRRT samples. The genomic profile of tumours reflected the known mutational landscape of PNET with MEN1 (34%), ATRX/DAXX (47%) alterations and a recurrent pattern of aneuploidy (38%) detected. A recurrent PSIP1::TBL1X fusion of unknown function was also identified in four tumours. The disease control rate following PRRT using RECIST1.1 and molecular imaging criteria was 88% (28/32). No mutational features were found to be statistically associated with progression-free survival. There was no significant increase in tumour mutational burden in the post-PRRT tumours, nor recurrent emergent mutational changes in cancer driver genes to explain progression to higher-grade disease, when observed. However, a small indel signature (ID8) previously associated with DNA damage repair by non-homologous end joining (NHEJ) was higher in PRRT-exposed compared with PRRT-naive samples (23.8 vs 4.8%, respectively; P < 0.001). Thus, comprehensive DNA analysis of pancreatic NETs did not identify biomarkers predictive of PRRT response nor evidence for high-level PRRT-induced genomic instability or hypermutation, yet mutation signature analysis supports NHEJ as being important for DNA repair and survival of neuroendocrine cells following exposure to beta-particle radiation.

Humans↗

PubMed2026

A CRISPR-Cas9 Toolkit Enabling Tunable Integration and Transient Homologous Recombination Enhancement in Yarrowia lipolytica.

Although the oleaginous yeast Yarrowia lipolytica is a promising microbial cell factory, its application remains constrained by inefficient homology-directed repair (HDR) and a lack of precise genomic integration tools. To address these limitations, we developed a comprehensive genetic toolkit featuring three synergistic advancements. First, we systematically identified 55 neutral integration sites with tunable expression profiles, enabling stable, position-independent gene integration with predictable transcriptional output across a 12.88-fold dynamic range. Second, we established a dual-readout high-throughput screening platform combining colony morphology analysis with hrGFP fluorescence. This approach accurately measures locus-specific homologous recombination (HR) efficiency while eliminating false positives by dominant non-homologous end joining (NHEJ). Third, we engineered a transient HR enhancement system by fusing the Sae2 exonuclease to Cas9 via a flexible (GGGGS)3 linker. This fusion significantly boosts HR efficiency and surpasses the cleavage activity of unmodified Cas9 without introducing permanent genomic modifications or compromising cellular fitness. Finally, HR efficiency for single-gene integration was increased from 46.5% to 77.5% while the dual-locus editing efficiency reached 64.1% when using 500-bp homology arms, and the engineered strains demonstrated improved genetic stability compared to those with constitutive HR enhancement.

Yarrowia↗

PubMed2025

SelectRepair Knockout: Efficient PTC-Free Gene Knockout Through Selectable Homology-Directed DNA Repair.

Generating nonessential gene knockouts using CRISPR/Cas9 technology is becoming increasingly common in biological research. In a typical workflow, the Cas9 endonuclease is used to induce a DNA double-strand break that relies on nonhomologous end-joining (NHEJ) to introduce a premature termination codon (PTC) in the target gene. The goal is to isolate clones in which the gene produces PTC-containing mRNA transcripts that are degraded via nonsense-mediated mRNA decay (NMD) to cause loss of gene function. Unfortunately, this approach is laborious, and not all PTCs trigger NMD. More importantly, mounting evidence suggest that PTC mutations can also result in a transcriptional adaptation response that can mask the effects of a PTC-mediated gene knockout. In this chapter, we present a PTC-free gene knockout strategy that implements homology-directed DNA repair (HDR) with selectable markers to substantially reduce the complexity of the screening and validation of genome edits in cells containing more than one gene copy as in the case of the commonly used hypotriploid HEK293 cell line. We describe how to obtain a complete knockout of the Ligase IV protein (LIG4) and provide considerations for the application of this SelectRepair Knockout method to other genes.

Humans↗

PubMed2025

Gene Editing and Protein Tagging in the Oomycete Phytophthora infestans Using CRISPR-Cas12a.

Molecular genetic tools such as CRISPR-Cas gene editing systems are invaluable for understanding gene and protein function and revealing the details of a pathogen's life and disease cycles. Here we present protocols for genome editing in Phytophthora infestans, an oomycete with global importance as a pathogen of potato and tomato. Using a vector system that expresses variants of Cas12a from Lachnospiraceae bacterium and its guide RNA from a unified transcript, we first present a method for editing genes through the non-homologous end-joining (NHEJ) pathway. We then describe an application of homology-directed repair (HDR), in which Cas12a is used to fuse a protein-coding gene with a fluorescent or epitope tag. Both methods should be adaptable to many oomycetes other than P. infestans.

Gene Editing↗

PubMed2026

Navigating nuclear space: How Rad51 filaments promote long-range homology search during homologous recombination - Lessons from budding yeast.

DNA double-strand breaks (DSBs) threaten genomic integrity, with erroneous repair leading to chromosomal rearrangements and pathologies. In eukaryotes, DSBs are primarily repaired via non-homologous end-joining (NHEJ) or homologous recombination (HR). HR restores genetic information by using an undamaged homologous sequence as a template, a process dependent on Rad51-mediated homology search. This review synthesizes recent advances in our understanding of HR, with a focus on the homology search process in mitotic cells, primarily using Saccharomyces cerevisiae as a model organism. We explore factors that limit recombination efficiency and discuss how Rad51 filament dynamics overcome spatial and temporal challenges imposed by nuclear architecture and chromatin dynamics, to ensure efficient HR. Key insights include the dynamic behavior of Rad51 filaments, which undergo cycles of compaction and extension, thereby optimizing exploration of the nuclear volume and increasing the likelihood of encountering distant homologous sequences. The interplay between long-range resection, filament elongation, and nuclear constraints further shapes the search process, balancing the need for extensive exploration with the risks of excessive DNA degradation and ectopic recombination. Collectively, these findings support an integrated model in which the efficiency and accuracy of homologous recombination are governed by regulated Rad51 filament dynamics and the constraints imposed by nuclear architecture.

Rad51 Recombinase↗

PubMed2026

Biallelic rescue of CTG18.1 in two Fuchs endothelial corneal dystrophy-derived iPSC lines (SCTCi047-A-2, SCTCi046-A-2) following a two-step gene editing strategy.

Fuchs endothelial corneal dystrophy (FECD) is an age-related condition distinguished by the degeneration of the corneal endothelium. An intronic CTG18.1 repeat in the transcription factor 4 (TCF4) gene has been associated with a 78-fold increased risk of developing the disease when at least one copy of the CTG18.1 expands above 50 repeats. Employing patient-derived material, we applied a dual CRISPR/Cas9-mediated editing approach to rescue the expansion. Combining non-homologous end-joining (NHEJ) and homologous direct repair (HDR) events, we generated two FECD-derived +/+(CTG)8 induced pluripotent stem cell (iPSC) lines, which were then successfully characterized, providing relevant isogenic controls for disease-modelling purposes.

Humans↗

PubMed2026

Ultra-sensitive profiling of CRISPR-Cas off-target effects with Tracking-seq2.

Accurate detection of off-target activity in primary human cells is crucial for ensuring the safety of gene therapies, yet existing methods often lack sufficient sensitivity. To address this limitation, we develop Tracking-seq2, an advanced technology that integrates exogenous 5' → 3' exonuclease treatment and non-homologous end joining (NHEJ) pathway inhibitors with the original Tracking-seq. Tracking-seq2 exhibits enhanced sensitivity in profiling off-target sites of diverse genome editors-including Cas9, Cas12a, cytosine base editors (CBEs), adenine base editors (ABEs), and prime editors (PEs). Critically, Tracking-seq2 is directly applicable to clinically relevant primary human cell types, such as T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs). Furthermore, our findings reveal that genomic variations drive distinct off-target heterogeneity across different individuals, highlighting the necessity for personalized safety assessment in clinical genome editing applications. Tracking-seq2 provides a robust platform for sensitive off-target detection in primary cells, with sensitivity comparable to or exceeding current state-of-the-art methods.

Humans↗

PubMed2021

Mechanism, cellular functions and cancer roles of polymerase-theta-mediated DNA end joining.

Cellular pathways that repair chromosomal double-strand breaks (DSBs) have pivotal roles in cell growth, development and cancer. These DSB repair pathways have been the target of intensive investigation, but one pathway - alternative end joining (a-EJ) - has long resisted elucidation. In this Review, we highlight recent progress in our understanding of a-EJ, especially the assignment of DNA polymerase theta (Polθ) as the predominant mediator of a-EJ in most eukaryotes, and discuss a potential molecular mechanism by which Polθ-mediated end joining (TMEJ) occurs. We address possible cellular functions of TMEJ in resolving DSBs that are refractory to repair by non-homologous end joining (NHEJ), DSBs generated following replication fork collapse and DSBs present owing to stalling of repair by homologous recombination. We also discuss how these context-dependent cellular roles explain how TMEJ can both protect against and cause genome instability, and the emerging potential of Polθ as a therapeutic target in cancer.

Animals↗

PubMed2025

SFPQ Promotes Homologous Recombination via mRNA Stabilization of RAD51 and Its Paralogs.

Double-strand break (DSB) repair occurs through non-homologous end joining (NHEJ) or homologous recombination (HR). To identify non-canonical factors that influence DSB repair outcomes, we parsed data from pooled genetic screens. Through this approach, we identified the splicing factor SFPQ, which has been previously reported to associate with DSBs and promote repair. Here, we show that SFPQ depletion alters DSB repair via HR. However, in contrast to other published work, we find that SFPQ does not localize to DSBs but instead stabilizes the expression of RAD51 and its paralogs independently of p53 activation or DNA damage. Our findings suggest that SFPQ contributes to constitutive DSB repair by maintaining RAD51 paralog mRNA stability rather than through direct interaction with DSBs or RAD51 protein and highlight indirect mechanisms by which RNA-binding proteins can influence genome stability.

DNA double-strand break repair (DSB repair)↗