Genome wide association study (GWAS) reports substantially outpace subsequent functional characterization. Pinpointing the causal effector gene(s) at GWAS loci remains challenging given the non-coding genomic residency of >98% of these signals. We previously implicated effector genes at GWAS loci for the complex and polygenic disorder of human insomnia using a high-resolution cell-specific, chromatin capture-based variant-to-gene mapping protocol, paired with sleep phenotyping in Drosophila . In this study, we leveraged a diurnal vertebrate model with higher genomic conservation, namely zebrafish, to screen our six highest confidence candidate genes and identify those whose loss-of-function impaired sleep characteristics related to human insomnia-like behaviors. Of these genes, we observed that CRISPR-mediated deletion of the zebrafish ortholog of MEIS1 produced nighttime specific sleep fragmentation and increased latency to sleep, pointing to a conserved role for MEIS1 in sleep maintenance. Comparing our human cell-based chromatin accessibility and contact maps with publicly available zebrafish spatial genomic data revealed highly conserved genomic architecture harboring the insomnia GWAS variant of interest. Notably, this genomic conservation was selective for the zebrafish ortholog which contributed to the sleep phenotype, meis1b, while the duplicated ohnolog meis1a proved dispensable. Motivated by this, we characterized the spatio-temporal expression of meis1b in zebrafish, showing it is comparable to human with respect to cerebellar granule progenitors. Ultimately, we found that loss of meis1b impairs cerebellar development. Together, our work provides a powerful model for screening human disorder risk genes for sleep fragmentation using a tractable vertebrate and supports a conserved cerebellar role for MEIS1 in sleep disturbance.

G protein-coupled receptors (GPCRs) that couple to the Gαq signaling pathway control diverse physiological processes, yet the full complement of cellular regulators for this pathway remains unknown. Here, we report the first genome-wide CRISPR knockout screen targeting a Gαq-coupled GPCR signaling cascade. Using a Drosophila model of adipokinetic hormone receptor (AkhR) signaling, we identified CG34449 (Zdhhc8) , encoding a palmitoyl acyltransferase and its adapter protein CG5447, as a top hit required for robust Gαq-mediated GPCR signaling. We show that Zdhhc8 enhances GPCR signaling through palmitoylation of Gαq, which promotes its membrane localization and function. Loss of Zdhhc8 markedly reduces palmitoylation of Gaq resulting in attenuation of AkhR/Gαq signaling and a reduction in receptor stability. Mechanistically, Zdhhc8 is necessary for palmitoylation of Gαq. These findings uncover Zdhhc8-dependent Gαq palmitoylation as a pivotal regulatory mechanism in GPCR signal transduction and highlight palmitoyl transferase as potential modulators of GPCR pathways.

Abstract 2.1 Background Adipogenesis is a highly organised series of events that facilitates the healthy expansion of adipose tissue, beginning during embryogenesis and continuing throughout life. White adipogenesis protects against lipotoxicity, influencing insulin resistance and obesity-related comorbidities. Brown adipogenesis enhances energy expenditure, thereby counteracting weight gain, lipotoxicity and insulin resistance. Recently, there has been a significant increase in interest regarding adipocyte differentiation, mainly focusing on the interplay between microRNAs (miRNAs) and the transcriptional cascade that governs adipogenesis and metabolic dysfunction. This study aimed to identify miRNAs regulating white and brown adipocyte differentiation and define miRNA action in a stem cell model of adipogenesis. 2.2 Methods Small RNAseq analysis of primary mouse brown and white adipocytes (WAs) identified miR-10b to be upregulated in mature brown adipocytes (BAs). We generated two model systems: 1) immortalized brown pre-adipocytes treated with miRNA inhibitors and 2) CRISPR/Cas9 KO of miR-10b in E14 mouse embryonic stem cells (mESCs). Both cell models were differentiated into mature adipocytes. To unravel the pathways that are affected by miR-10b depletion, a transcriptomic analysis was performed at key time points. 2.3 Results Both cell models showed that miR-10b-5p depletion severely impaired differentiation into mature adipocytes, as indicated by a lack of lipid droplet formation and reduced adipogenic gene expression. Gene expression analysis supports that miR-10b-5p directs embryonic stem (ES) cells towards the mesoderm lineage, promoting commitment to pre-adipocytes by downregulating Gata6 and its downstream target Bmp2. This mechanism appears to be unaffected in BAs. Our study demonstrated that miR-10b-5p regulates the later stages of adipogenesis, at least in part, by downregulating Tub, a direct target of miR-10b-5p. We also confirmed that miR-10b-5p alleviated the halted differentiation phenotypes of adipocytes by supressing the G Protein Signalling pathway mediated by Tubby. 2.4 Conclusions These results evidence that miR-10b inhibition plays a dynamic role in adipocyte biology, as its inhibitory effects manifest differently during the stem cell preadipocyte proliferation state and during the maturation phase of adipocytes. Collectively, our study demonstrated that miR-10b-5p may represent a new potential therapeutic target for lipodystrophy and obesity.

Abstract Background DOT1L, a histone H3 lysine 79 (H3K79) methyltransferase, is a potential therapeutic target in various malignancies. In the present study, we aimed to clarify the antitumor effect of DOT1L inhibition in breast cancer. Methods Estrogen receptor (ER)-positive/HER2-negative breast cancer cells (MCF7) and ER-negative/HER2-positive cells (SKBR3) were treated with a DOT1L inhibitor (SGC0942, EPZ-5676), after which colony formation assays, cell cycle assays, flow cytometry, gene expression microarray analysis, chromatin immunoprecipitation sequencing (ChIP-seq) and single-cell Assay for Transposase-Accessible Chromatin sequencing (scATAC-seq) were performed. Genetic ablation of STING was performed using the CRISPR/Cas9 system. Results Treatment with a DOT1L inhibitor suppressed proliferation and induced cell cycle arrest and apoptosis in both ER-positive/HER2-negative and ER-negative/HER2-positive cells. Transcriptome and epigenome analysis revealed that DOT1L inhibition activated transcription of a number of interferon (IFN)-related genes (IRGs) in breast cancer cells. We also found that DOT1L inhibition upregulated type I and type III IFNs and cell surface human leukocyte antigen (HLA) class I expression. Notably, DOT1L inhibition induced DNA damage and upregulated levels of cytoplasmic DNA in breast cancer cells. CRISPR/Cas9-mediated knockout of STING in breast cancer cells significantly suppressed the IFN signaling activated by DOT1L inhibition and attenuated the antitumor effects. Moreover, scATAC-seq analysis revealed that DOT1L inhibition suppressed expression of ERBB2 in HER2-positive breast cancer cells. Conclusions These findings suggest that the anti-breast cancer cell effects of DOT1L inhibition are mediated by multiple mechanisms, including activation of innate immune signaling.

Membrane protection against oxidative insults is achieved by the concerted action of glutathione peroxidase 4 (GPX4) and endogenous lipophilic antioxidants such as ubiquinone and vitamin E. Deficiencies in these protective systems lead to an increased propensity to phospholipid peroxidation and ferroptosis. More recently, ferroptosis suppressor protein 1 (FSP1) was identified as a critical ferroptosis inhibitor acting via regeneration of membrane-embedded antioxidants. Yet, regulators of FSP1 are largely uncharacterised, and their identification is essential for understanding the mechanisms buffering phospholipid peroxidation and ferroptosis. Here, we conducted a focused CRISPR-Cas9 screen to uncover factors influencing FSP1 function, identifying riboflavin (vitamin B₂) as a new modulator of ferroptosis sensitivity. We demonstrate that riboflavin, unlike other vitamins that act as radical-trapping antioxidants, supports FSP1 stability and the recycling of lipid-soluble antioxidants, thereby mitigating phospholipid peroxidation. Furthermore, we show that the riboflavin antimetabolite roseoflavin markedly impairs FSP1 function and sensitises cancer cells to ferroptosis. Thus, we uncover a direct and actionable role for riboflavin in maintaining membrane integrity by promoting membrane tolerance to lipid peroxidation. Our findings provide a rational strategy to modulate the FSP1-antioxidant recycling pathway and underscore the therapeutic potential of targeting riboflavin metabolism, with implications for understanding the interaction of nutrients and their contributions to a cell’s antioxidant capacity.

ABSTRACT Ferroptosis, a regulated form of cell death driven by excessive lipid peroxidation, has emerged as a promising therapeutic target in cancer. Ferroptosis suppressor protein 1 (FSP1) is a critical regulator of ferroptosis resistance, yet the mechanisms controlling its expression and stability remain mostly unexplored. To uncover regulators of FSP1 abundance, we conducted CRISPR-Cas9 screens utilizing a genome-edited, dual-fluorescent FSP1 reporter cell line, identifying both transcriptional and post-translational mechanisms that determine FSP1 levels. Notably, we identified riboflavin kinase (RFK) and FAD synthase (FLAD1), enzymes which are essential for synthesizing flavin adenine dinucleotide (FAD) from vitamin B2, as key contributors to FSP1 stability. Biochemical and cellular analyses revealed that FAD binding is critical for FSP1 activity. FAD deficiency, and mutations blocking FSP1-FAD binding, triggered FSP1 degradation via a ubiquitin-proteasome pathway that involves the E3 ligase RNF8. Unlike other vitamins that inhibit ferroptosis by scavenging radicals, vitamin B2 supports ferroptosis resistance through FAD cofactor binding, ensuring proper FSP1 stability and function. This study provides a rich resource detailing mechanisms that regulate FSP1 abundance and highlights a novel connection between vitamin B2 metabolism and ferroptosis resistance with implications for therapeutic strategies targeting FSP1 in cancer.

ABSTRACT Escherichia coli ( E. coli ) is a common bacterium in the human gut and an important cause of intestinal and extraintestinal infections. Some E. coli sequence types (ST) are associated with high pathogenicity. The Extraintestinal Pathogenic E. coli (ExPEC) ST131 is a globally distributed multidrug-resistant human pathogen associated with urinary tract and bloodstream infections. Antibiotic-resistant infections often lead to antibiotic treatment failure, underscoring the need of developing alternative treatments. The highly selective antimicrobial potential of CRISPR-Cas9 has been demonstrated in a range of model organisms. However, the effectiveness of CRISPR-Cas9 in combating ST131-associated infections and the consequences of CRISPR-Cas9 treatment, such as the emergence of escapers, remains unclear. Here, we investigated the antimicrobial activity of CRISPR-Cas9 against ST131 and assessed the frequency and genetic basis of escape. We conjugatively delivered CRISPR-Cas9 to ST131 isolates which carried cefotaxime-resistance-encoding target gene bla CTX-M-15 in the chromosome and characterized escape subpopulations. Two main types of escapers emerged: bla CTX-M-15 -positive escapers carried dysfunctional CRISPR-Cas9 systems and arose at a ∼10 −5 frequency. Instead, bla CTX-M-15 -negative escapers presented chromosomal deletions involving bla CTX-M-15 loss. The frequency of bla CTX-M-15 loss depended on the bla CTX-M-15 genetic context. Specifically, bla CTX-M-15 -negative escapers emerged at low frequency (∼10 −5 ) in isolates where bla CTX-M-15 was located downstream of insertion sequence (IS) IS Ecp1 , while escapers emerged with high frequency (∼10 −3 ) in isolates where bla CTX-M-15 was flanked by IS 26 . This work emphasizes how the genetic context of target genes can drive the outcome of CRISPR-Cas9 tools, where the presence of IS 26 may drive increased frequencies of escape. IMPORTANCE In the past decade CRISPR-Cas9 has emerged as an efficient antimicrobial tool capable of selective elimination of targeted bacteria. Even though it has been well described that bacteria can evolve to escape targeting by CRISPR-Cas9, the mechanisms of bacterial escape and their consequences remain largely elusive. In this study, we demonstrate the antimicrobial efficacy of CRISPR-Cas9 against natural isolates of Escherichia coli ST131, a clinically relevant pathogen, and elucidate the mechanism of escape from antimicrobial activity. We identify two distinct mechanisms of escape, which involve either dysfunctional CRISPR-Cas9 activity, or loss of the target gene ( bla CTX-M-15 ), with the latter occurring at frequencies that depend on the genetic context of the target gene. These findings provide important insights into the frequency and mechanisms of bacterial escape from CRISPR-Cas9-based antimicrobials and offer a foundation for the development of more effective treatments.

LMX1B, a LIM-homeodomain family transcription factor, plays critical roles in the development of multiple tissues, including limbs, eyes, kidneys, brain, and spinal cord. Mutations in the human LMX1B gene cause the rare autosomal-dominant disorder, Nail-patella syndrome which affects development of limbs, eyes, brain, and kidneys. In zebrafish, lmx1b has two paralogues: lmx1ba and lmx1bb. While lmx1b morpholino data exists, stable mutants were previously lacking. Here we describe the characterisation of lmx1b stable mutant lines, with a focus on development of tissues which are affected in Nail-patella syndrome. We demonstrate that the lmx1b paralogues have divergent developmental roles in zebrafish, with lmx1ba affecting skeletal and neuronal development, and lmx1bb affecting renal development. The double mutant, representing loss of both paralogues (lmx1b dKO) showed a stronger phenotype which included additional defects to trunk muscle patterning, and a failure to fully inflate the notochord leading to a dramatic reduction in body length. Overall, these mutant lines demonstrate the utility of zebrafish for modelling Nail-patella syndrome and describe a previously undescribed role for lmx1b in notochord cell inflation.

ABSTRACT Recent breakdowns in the supply chains of antiretroviral therapy (ART) to lower income countries, where they are needed most, underpin the pressing need for an accessible and scalable HIV cure that would allow ART-free control of the virus. The ‘shock-and-kill’ cure concept shows promise in its ability to reactivate HIV-1 transcription in latently infected cells in vivo in people living with HIV-1 but has failed to result in a meaningful reduction of the size of the reactivatable viral reservoir. We therefore hypothesised that the efficiency of reversal of transcriptional quiescence, and the resulting HIV-1 antigen expression and presentation, may be insufficient to achieve full immunological visibility of HIV-1-infected cells. To gain a deeper understanding of potential LRA-specific shortcomings, we developed a novel model of HIV-1 latency - Jurkat E6.1 subclonal cell lines, each harbouring a single, full-length, NL4.3-based provirus with a GFP OPT reporter inserted into the V5 loop of the viral Env protein. Using this model, we quantified HIV-1 reactivation, as well as expression and surface presentation of Env after treatment with a panel of known latency reversal agents (LRAs) and their synergistically-acting combinations. HIV-1 reactivation with the PKC agonist Bryostatin-1 limited the cell-surface presentation of viral Env, a phenomenon we found being linked to Bryostatin-1’s ability to induce the expression of the restriction factor GBP5 in T-cell lines and primary CD4+ T-cells, and induced a cellular state which was resistant to apoptosis. A combined treatment of Bryostatin-1 and the BET inhibitor (iBET) JQ1 resulted in synergistic HIV-1 reactivation and boosted levels of cell-surface Env, but failed to reduce resistance to apoptosis. In contrast, the combination of the SMAC mimetic (SMACm) AZD5582 and JQ1 markedly boosted cell-surface Env levels, without co-induction of GBP5, T-cell activation or apoptotic resistance. Crucially, HIV-1 Nef was found to be a potent antagonist of the cytotoxic killing of reactivating cells, most likely by its ability to inhibit apoptosis. Nef knockout, however, when paired with the AZD/JQ1 combination, displayed highest potency in inducing immune-mediated elimination of latently infected T-cells and presents a promising new approach for HIV cure programs.

The identification of novel antimalarials with activity against both the liver and blood stages of the parasite lifecycle would have the dual benefit of prophylactic and curative potential. However, one challenge of leveraging chemical hits from phenotypic screens is subsequent target identification. Here, we use in vitro evolution of resistance to investigate nine compounds from the Tres Cantos Antimalarial Set (TCAMS) with dual liver and asexual blood stage activity. We succeeded in eliciting resistance to four compounds, yielding mutations in acetyl CoA synthetase (AcAS), cytoplasmic isoleucine tRNA synthetase (cIRS), and protein kinase G (PKG) respectively. Using a combination of CRISPR editing and in vitro activity assays with recombinant proteins, we validate these as targets for TCMDC-125075 (AcAS), TCMDC-124602 (cIRS), and TCMDC-141334 and TCDMC-140674 (PKG). Notably, for the latter two compounds, we obtained a T618I mutation in the gatekeeper residue of PKG, consistent with direct interaction with the active site, which we modelled with molecular docking. Finally, we performed cross-resistance evaluation of the remaining five resistance-refractory compounds using the Antimalarial Resistome Barcode sequencing assay (AReBar), which examined a pool of 52 barcoded lines with mutations covering >30 common modes of action. None of the five compounds where in vitro evolution of resistance was not successful yielded validated hits using AReBar, indicating they likely act via novel mechanisms and may be candidates for further exploration.

Here we present pCASKD, a single-plasmid system for scarless chromosomal editing in Escherichia coli . Our plasmid pCASKD integrates CRISPR-Cas9-mediated counterselection, Lambda-Red recombineering, and temperature-sensitive plasmid curing into a 12 Kb vector to enable kilobase-scale insertions and deletions using a linear dsDNA donor and homologous recombination. Using the flagellar stator motAB locus as a model, we demonstrate that pCASKD enables efficient knock-in and knock-out edits with lower donor DNA input and reduced false positives compared with its parent, multi-plasmid system, No-SCAR. Using a single plasmid reduces transformation steps, accelerates screening, and increases the frequency of correctly edited clones. The protocol can be completed in five days, with potential for further optimization, offering a compact and efficient alternative for microbial genome engineering.

ABSTRACT Site-directed RNA editing, especially RNA base editing, allows for specific manipulation of RNA sequences, making it a useful approach for the correction of pathogenic mutations. Correction of RNA transcripts allows therapeutic gene editing in a safe and reversible manner and avoids permanent alterations in the genome. RNA-targeting CRISPR-Cas nucleases (e.g., CRISPR-Cas13) enable delivery within a single adeno-associated virus (AAV) vector for RNA base editing, making the approach clinically feasible. Here, we used the inactive CRISPR-Cas13bt3 (also known as Cas13X.1) fused to the ADAR2 deaminase domain (ADAR2DD) for targeted correction of inherited retinal disease (IRD) mutations. First, we show in vitro that dCas13bt3-ADAR2DD can efficiently correct a pathogenic nonsense mutation (c.130C>T [p.R44X]) found in the mouse Rpe65 gene and recover protein expression in retinal pigment epithelium cells (RPEs). Across clinically reported RPE65 mutations, we observed editing efficiencies ranging from 0% to 60%. In the Rpe65 -deficient mouse model of retinal degeneration (rd12), we observed that RNA base editing can recover Rpe65 expression in RPEs and rescue retinal function with no observable adverse effects. We further employed our RNA base editor against the large USH2A gene to assess the promise of RNA base editing for addressing untreatable IRDs caused by genes too large for AAV gene delivery. Against the human USH2A in vitro , we observed up to 60% on-target efficiency. We further found that gRNA mismatches, domain-inlaid ADAR2DD design and nucleocytoplasmic shuttling of the RNA base editor optimised on-target and bystander editing for a highly precise base editor. Against the mouse Ush2a in vitro , we similarly observed up to 60% on-target editing in mammalian cells, while in the Ush2a W3947X mice, we observed ∼12% on-target editing, with no impact on retinal structure or function, or transcriptome-wide editing. Overall, our findings demonstrate dCas13bt3-ADAR2DD as a potent tool for gene therapy against IRDs, addressing a significant unmet clinical need in ophthalmology.

Some phages have evolved the ability to cooperate to evade the immunity triggered by their bacterial host. A first exposure to the phage may weaken the host defences and allow later infections to be successful. Because this cooperation requires sequential infections, the phage can invade the host population only if its initial density is sufficiently high in a well-mixed environment. However, most natural bacterial populations are spatially structured. Could spatial structure create more favourable conditions for viral spread? Here we study the effect of spatial structure on the dynamics of cooperative anti-CRISPR (Acr) phages spreading in a population of CRISPR-Cas resistant Pseudomonas aeruginosa bacteria. We show experimentally that spatial structure does not always promote the spread of Acr-phages. In particular, the effect of spatial structure is modulated by the efficacy of the bacterial host’s CRISPR-Cas resistance and by the efficacy of the phage Acr protein. These results are discussed in the light of a mathematical model we developed to describe the spread of the phage. The model allows us to understand the ambivalent effects of spatial structure via its effects on the reproduction and on the persistence of the phage. More generally, we find that spatial structure can have opposite effects on the epidemiological dynamics of the phage, depending on the properties of the Acr protein encoded by the phage. This joint experimental and theoretical work yields a deeper understanding of the spatial dynamics of cooperative strategies in phages.

Understanding the gene regulatory mechanisms underlying brain function is crucial for advancing knowledge of the genetic basis of neurologic diseases. Cis-regulatory elements (CREs) play a pivotal role in gene regulation, and their evolutionary conservation can offer valuable insights. Importantly, the function and evolution of CREs are affected not only by primary sequence, but also by the cis - and trans -regulatory context. However, comparative functional analyses across species have been limited, leaving how these regulatory landscapes evolve in the brain largely unresolved. Here, we generated single-nucleus multiomic (snRNA- and snATAC-seq) data from cortex tissue across nine mammalian species and identified candidate CREs (cCREs) in a cell type–specific manner. We developed a multidimensional framework of conservation to assess sites of shared function that integrates sequence, chromatin accessibility, and enhancer–gene associations. Using massively parallel reporter assays (MPRA) in human neural progenitor cells and neurons, we measured activity of cCREs including both conserved and human-specific regions. CRISPR interference validated conserved enhancer function, including at neurodevelopmentally important genes like FAM181B . Motif enrichment identified transcription factors distinguishing conserved versus recent cCREs. Linkage disequilibrium score regression indicated that both conserved and human-specific cCREs were enriched for neuropsychiatric GWAS risk, while neurodegenerative risk was confined to conserved elements. Our findings define functional dimensions of enhancer conservation and demonstrate how regulatory evolution shapes human brain biology and disease susceptibility.

Genetic interactions are typically studied by looking at the phenotype that results from disruption of pairs of genes, as well as from higher order combinations of perturbations. Systematically interrogating all pairwise combinations provides insights into how genes are organized into pathways and complexes to sustain cellular homeostasis and how interacting genes respond to stressors and external signals. Genetic interactions have been studied extensively in yeast, due, in part, to the availability of a systematic collection of gene knockouts, and the development of Synthetic Genetic Array (SGA) technology. In contrast, such approaches are more challenging in human cells and therefore comparable data for human cells is scarce. This study introduces an innovative approach to functionally characterize genetic interactions in human cells through CRISPR/Cas9 screens using a pooled genome-wide knockout library in NALM6 cells. By combining a single guide RNA (sgRNA) targeting the gene of interest (aka the query) in cells already infected with an inducible genome-wide sgRNA pool, it is possible to achieve near saturation of genome-wide double knockouts. We conducted 26 of these screens, which we term “gene by genome-wide” knockout screens. This approach can be rapidly performed, in part, because it bypasses the need to generate genotyped isogenic knockout clones. Data from these screens identified both expected and novel synthetic lethal and synthetic rescue interactions, demonstrating that this strategy is effective for large-scale genetic research in human cells. Additionally, we show that these GBGW screens can be combined with chemical perturbation to reveal new synthetic interactions that are not apparent without drug treatment. Finally, we show that cDNA overexpression can be incorporated with genome-wide knockouts to systematically explore gain-of-function scenarios. The complete dataset is accessible on the ChemoGenix website (URL: https://chemogenix.iric.ca ).

Human papillomavirus (HPV) is a major etiological factor in cervical, anal, and oro-pharyngeal cancers. Although prophylactic vaccines have substantially reduced infec-tion rates, effective therapeutic options for established HPV-associated malignancies remain limited. This review provides an up-to-date overview of emerging strategies to treat HPV-driven tumors. Key approaches include immune checkpoint inhibitors, therapeutic vaccines such as VGX‑3100 and PRGN‑2012, and gene-editing tools like CRISPR/Cas9. Epigenetic drugs, particularly histone deacetylase inhibitors, show promise in reactivating silenced tumor suppressor genes and enhancing antitumor immunity. In addition, natural bioactive compounds and plant-derived molecules are being explored as complementary anti-HPV agents, while drug repurposing and combination therapies offer cost-effective opportunities to broaden treatment options. We also highlight the role of patient-derived organoid models as powerful platforms for personalized drug screening and functional assessment. By integrating these therapeutic innovations with precision oncology approaches, this review outlines a multi-dimensional framework aimed at improving clinical outcomes and quality of life for patients with HPV-associated cancers.

Terpenoids, vital pharmaceutical compounds, face production challenges due to low yields in native plants and ecological concerns. This review synthesizes recent advances in metabolic engineering strategies implemented across three complementary platforms: native medicinal plants, microbial systems, and heterologous plant hosts. We elucidate how the "Genomic Insights to Biotechnological Applications" paradigm, empowered by multi-omics technologies such as genomics, transcriptomics, metabolomics, etc., drives research advancements. These technologies facilitate the identification of key biosynthetic genes and regulatory networks. CRISPR-based tools, enzyme engineering, and subcellular targeting are highlighted as transformative strategies. Significant yield improvements have been demonstrated, with artemisinin and paclitaxel precursors showing considerable increases in production through strategic co-expression and optimization techniques. Persistent challenges such as metabolic flux balancing, cytotoxicity, and scale-up economics are discussed alongside emerging solutions including machine learning and photoautotrophic chassis. We conclude by outlining a roadmap for industrial translation that emphasizes the critical integration of systems biology and synthetic biology approaches to accelerate the transition of terpenoid biomanufacturing from discovery to commercial scale.

Genome editing technologies including CRISPR/Cas9, TALENs, and ZFNs offer a unique opportunity to eradicate HPV by directly disrupting its oncogenes E6 and E7, thereby restoring tumour‑suppressor pathways. In this review, we provide a comprehensive overview of recent advances in HPV‑targeted genome editing, summarising key preclinical findings that demonstrate tumour regression and apoptosis in HPV‑positive models, as well as the first‑in‑human clinical trials assessing safety and feasibility of local CRISPR‑based therapies. We also compare the relative strengths and limitations of each editing platform, discuss delivery strategies, and highlight their potential integration with immunotherapy and standard cancer treatments. While genome editing shows unprecedented precision and durability in targeting viral oncogenes, challenges such as efficient delivery, minimising off‑target effects, and navigating regulatory and ethical considerations remain. Continued optimisation of high‑fidelity nucleases, tissue‑specific delivery vehicles, and personalised guide design will be essential to translate these promising approaches into routine oncology practice. Genome editing thus represents a paradigm shift in HPV therapy, with the potential to transform management of both persistent infections and established cancers.

DNASE1L3 is a key endonuclease, essential for proper fragmentation and clearance of cell-free DNA (cfDNA). The p.R206C common variant impairs DNASE1L3 secretion and activity, causing aberrant cfDNA fragmentation and therefore affecting liquid biopsy-based screening and diagnostics. Existing studies on DNASE1L3 relied on resource-intensive murine models or plasmid-based overexpression, which do not accurately represent native expression. To address this, we developed an isogenic HEK293T cell line model by using CRISPR Prime Editing for endogenous expression of DNASE1L3 R206C . We analyzed the cfDNA composition directly from conditioned culture medium and found that fragment size distributions in mutant cells mimics the hypofragmented profiles previously observed in plasma samples from p.R206C carriers. We also showed that in vitro treatment of hypofragmented cfDNA with recombinant wildtype DNASE1L3 could enrich for mononucleosomal fragments, with fragment end-motifs characteristic of DNASE1L3 cleavage activity. This could open avenues for DNASE1L3 as a candidate pre-treatment agent to improve the accuracy and efficiency of cfDNA sequencing-based diagnostics in hypofragmented liquid biopsies. These findings demonstrate that our isogenic cell line model provides a controlled system to study cfDNA fragmentation biology and DNASE1L3 function.

Long-term recurrence in breast cancer is driven by reactivation of dormant disseminated tumour cells (DTCs) and remains a major clinical challenge, particularly in oestrogen receptor positive (ER⁺) tumours. This process is underpinned by regulation of the cell cycle machinery that controls quiescence maintenance and exit. HES1, a Notch pathway transcription factor, regulates key cell cycle genes and has been shown to demonstrate protein expression oscillations. Here, we sought to establish whether HES1 oscillations may regulate ER+ cancer cell quiescence and reactivation. To investigate this, we developed a fundamental in vitro model of cell cycle arrest and re-entry based on reversible CDK4/6 inhibition (CDK4/6i) with palbociclib, compatible with quantitative single-cell live-imaging of a knock-in endogenous HES1 reporter. Consistent with earlier findings, HES1 exhibited ∼24-hour protein oscillations in cycling cells demonstrating a reproducible dip in protein expression prior to S-Phase. During CDK4/6i-mediated arrest, the ∼24h HES1 oscillation was lost, HES1 levels were maintained at a moderately higher level and HES1 exhibited smaller dips. Similar changes were observed in unperturbed, spontaneously quiescent cells. Following release from CDK4/6i and cell cycle re- entry, these alterations were reversed and the characteristic G1/S HES1 dip was observed. Preventing this dip at the point of release, by inducibly sustaining HES1 with a Tet-On system, upregulated the cell cycle inhibitor p21, impeded cell cycle re-entry and induced cell death. These findings suggest that manipulating HES1 dynamics could represent a promising therapeutic approach to prevent reactivation of dormant tumour cells. Significance Statement Breast cancer can recur years after initial treatment due to reactivation of dormant tumour cells. Understanding how these cells exit dormancy is crucial for preventing relapse. We investigated HES1, a transcription factor with rhythmic protein oscillations, and its role in regulating quiescence in oestrogen receptor-positive (ER⁺) breast cancer cells. Using live-cell imaging and a reversible cell cycle arrest model, we show that HES1 dynamics change during dormancy and reactivation, and that disrupting these oscillations prevents cell cycle re-entry and induces cell death. These findings reveal HES1 protein dynamics as a potential therapeutic vulnerability and highlight a novel strategy to target dormant cancer cells to prevent their reactivation.

CRISPR-Cas9 has revolutionized plant genome editing by enabling precise introduction of insertion/deletion (indel) mutations, critical for functional genomics and crop improvement studies. Sanger sequencing, combined with bioinformatics tools like the INDIGO webserver from Gear Genomics, is essential for validating these mutations. However, manual analysis of large numbers of Sanger sequencing (.ab1) files is labor-intensive, particularly when analyzing multiple guide RNA (gRNA) target regions. We developed a Python-based automation pipeline using Selenium with integrated highlighting of protospacer adjacent motif (PAM) regions in the resulting HTML reports. This pipeline enhances scalability of Sanger sequence data analysis and improves result interpretability by automating PAM region identification and supporting multiple gRNA regions. This tool significantly accelerates CRISPR-Cas9-mediated mutation analysis, offering a high throughput, reproducible solution for genome editing research.

Proteolysis-Targeting Chimeras (PROTACs) and Molecular Glue Degraders (MGDs) canonically target proteins for degradation by recruiting them to a single E3 ligase complex. While heterotrivalent PROTACs that can co-opt multiple E3 ligase complexes have been described, to our knowledge all MGDs reported to date are dependent on a single E3. Here, using orthogonal genetic screening, biophysical and structural analyses, we show that a monovalent MGD can covalently recruit CUL4DCAF16 and CRL1FBXO22 in a parallel and redundant manner to degrade SMARCA2/4. Deep mutational scanning identifies a single cysteine (Cys173) in DCAF16 essential for degrader activity, and intact protein MS confirms covalent adduct at this site. The cryo-EM structure of the DCAF16:SMARCA2:degrader ternary complex reveals a unique binding mode and a distinct interface of neo-interactions, providing insights into degrader specificity. We demonstrate that E3 ligase dependency can be tuned both chemically and genetically. Minimal alterations to the compound's "degradation tail" switches ligase preference from DCAF16 to FBXO22, while a single L59W mutation on DCAF16 is sufficient to drive DCAF16 engagement for otherwise FBXO22-dependent compounds. These results establish a molecular and structural framework for the design of tuneable dual glue degraders that could mitigate challenges from resistance mechanisms in degrader therapies.

ABSTRACT Efficient utilization of complex biomass-derived sugars and tolerance to inhibitors are key requirements for the viability of lignocellulosic-based biorefineries. In this study, a two-stage evolution of an industrial yeast strain engineered with a xylose isomerase pathway yielded strain AceY.14, which exhibited improved fermentative performance and increased tolerance to acetic acid. Whole-genome sequencing of the evolved strain identified SNPs in ZWF1 , a component of the pentose phosphate pathway (PPP), and in the G1 cyclin gene CLN3 , both of which were functionally validated through CRISPR and reverse engineering. The zwf1 E191D mutation reduced xylitol accumulation, alleviating inhibition of xylose isomerase and enhancing flux through the non-oxidative branch of the PPP, while the frameshift cln3 T556fs mutation unexpectedly improved acetic acid tolerance and xylose consumption in the evolved strain, also affecting cell size and growth. Genome sequencing of AceY.14 also revealed a significant reduction in the xylA gene copy number, likely decreasing the metabolic burden associated with high xylose isomerase expression. A synergistic effect was observed in the isu1 Δ /zwf1 Δ double mutant, further boosting xylose consumption rates. A diploid derivative (AceY-2n) demonstrated high productivity and robustness in fermentations using hydrolysates from various lignocellulosic feedstocks, highlighting the strain’s potential for industrial-scale applications. These findings reveal novel metabolic targets for strain optimization and offer valuable insights for the rational engineering of yeast platforms for sustainable biofuel and bioproduct production.

SUMMARY During development, many cell types transition from canonical to non-canonical cell cycles, such as endomitosis and endoreplication, in which they duplicate their DNA but do not divide, giving rise to polyploidy. Little is known on the regulation of endomitosis, where cells enter M phase, but do not perform cytokinesis. Here, we investigate how cells initiate and execute endomitosis in the C. elegans intestine and find that endomitotic cells fail to assemble a central spindle or initiate cytokinetic furrowing. We find that endomitotic cells transcriptionally repress multiple cytokinesis regulators, especially the central spindle factors ZEN-4 Mklp 1 , CYK-4 RacGap 1 and SPD-1 Prc 1 . Intestinal cells lose the capacity to perform cytokinesis in late embryogenesis, and we find that the conserved DREAM (DP, RB-related, E2F and MuvB) complex is involved in the repression of central spindle genes. Together, our work demonstrates that the transition to endomitosis is a well-defined switch that relies on the transcriptional repression of cytokinesis genes.

Engineering animal models with self-sustained luminescence could enable non-invasive longitudinal monitoring of molecular events in living animals. To create animal models that report physiology with autoluminescence, both luciferin biosynthesis enzymes and the luciferase need to be optimised. Previous work on engineering the autoluminescence pathway from fungi resulted in the development of nnLuz_v3, a version of Neonothopanus nambi luciferase with enhanced thermal stability. Here, we generated an nnLuz_v3 reporter of endogenous Cyp1a1 expression as a measure of aryl hydrocarbon receptor (AHR) activation, assessing the performance of nnLuz_v3 in vivo at physiologically relevant expression levels. As AHR dynamically responds to metabolic, environmental and dietary changes it provides a validated platform to assess novel luminescence approaches. In Cyp1a1-nnLuz mice bioluminescence signal was stable, allowing the generation of well-resolved luminescence images both on standard in vivo imaging equipment and consumer-grade cameras. Using mice and nematode models, we demonstrated limited oral availability of the fungal luciferin, potentially compatible with delivering the substrate via food or the microbiome. Our results are an encouraging first step in the generation of an autoluminescent mammalian model of a molecular event and encourage optimisation of other enzymes of the fungal luciferase pathway.