I present \textsc{SEQUENTION}, a timeless theoretical framework for biological change in which the living biosphere is a three-dimensional shadow of a complete four-dimensional counterspace holding the full content of viable genotype--phenotype--environment relations. In this view, ontic time does not exist; what is commonly described as ``evolution through time'' is a foliation artifact of admissible projections from a unified 4-D content field. I formalize \textsc{SEQUENTION} with an \emph{extrinsic constitutive law} that maps informational gradients to observable fixation and trait-change fluxes via a single embedding scale (\aD). Classical population-genetic dynamics emerge as \emph{gauge choices} in a high-constraint limit. % === AMENDED v2.0 === The term "falsifiable predictions" is a categorical error, as A3 is non-falsifiable. Replaced. I derive a program of \textbf{cartographic inquiries}---curvature invariants for convergent adaptations, order-invariant terminal phenotypes within projection cones, slice-invariant developmental complexity, corridor-governed macroevolutionary bursts without temporal rates, and protocol-independent invariants in laboratory evolution---and provide protocols (deep mutational scanning, modular CRISPR assays, comparative morphometrics, microbial evolution) to \textbf{map them}. Recasting evolution as projection geometry rather than temporal process, I aim to unify convergence, canalization, and punctuated patterns under a single, testable law. Within \textsc{SEQUENTION}, \emph{uncertainty, randomness, and probability} have no ontic status; they are artifacts of foliation and incomplete conditioning.

Most terrestrial animals exhibit narrow salinity tolerance compared to their marine counterparts. Previous studies identified osm-11 (which encodes a Notch co-ligand) mutations as a driver of hyper-saline tolerance in Caenorhabditis elegans , but mechanistic insights remained unclear. This study employs RNA sequencing and CRISPR/Cas-9 genome editing to demonstrate that osm-11 mutations enhance salinity stress resistance through up-regulation of fatty acid metabolism ( acdh-12 , acs-17 ) and cytochrome P450 pathways ( ugt-15 ), while suppressing calcium signaling. Furthermore, we demonstrated that acdh-12 mutation impairs salinity-stress tolerance by activating ferroptosis and mitophagy, accompanied by down-regulated oxidative phosphorylation and up-regulated autophagic pathways. Morphological observations show that mitochondrial fragmentation contributes to wild-type nematode mortality under high salinity, while enlarged lipid droplets in wild-types correlate with reduced β-oxidation gene expression ( dhs-28 , daf-22 ), whose knockout disrupts tolerance in mutants. These findings unravel the multi-pathway regulatory network of osm-11 -mediated salinity tolerance, providing mechanistic insights for developing protective strategies against environmental salinity stressors impacting animal survival.

ABSTRACT In classic disease models, removing a pathological insult restores homeostasis. Yet, addiction persists far beyond the period of active drug use. Cocaine abstinence induces changes in gene expression and neuronal signaling in reward-related brain regions that limit recovery during abstinence. We found that 2 weeks of abstinence increased Cartpt (cocaine- and amphetamine-regulated transcript) in the mouse nucleus accumbens and decreased repressive H3K27me3 at the Cartpt locus. While endogenous CART peptide is best described for its anorexigenic function, it is also implicated in human addiction and dopamine homeostasis. To test the causal relevance of Cartpt chromatin remodeling, we used CRISPR-based epigenetic editing tools, dCas9-FOG1 and dCas9-JMJC-ZF, to manipulate H3K27me3 at Cartpt in vivo . Enriching H3K27me3 in D1 neurons repressed Cartpt expression and augmented acquisition and extinction of cocaine preference. These results show that CRISPR epigenetic editing can recapitulate endogenous chromatin states to modulate addiction-related behavior, highlighting broad therapeutic potential of both Cartpt and epigenetic editing. Abstract Figure

Abstract Methyltransferase PRC2 (Polycomb Repressive Complex 2) deposits histone H3K27 trimethylation to establish and maintain epigenetic gene silencing. PRC2 is precisely regulated by accessory proteins, histone post-translational modifications, and, particularly, RNA. Research on PRC2-associated RNA has mostly focused on the tight-binding G-quadruplex (G4) RNAs, which inhibit PRC2 enzymatic activity in vitro and in cells, a mechanism explained by our recent cryo-EM structure showing G4 RNA-mediated PRC2 dimerization. However, PRC2 binds a wide variety of RNA sequences, and it remained unclear how diverse RNAs beyond G4 associate with and regulate PRC2. Here, we show that variations in RNA sequence elicit disparate effects on PRC2 function. A G-rich RNA lacking consecutive G’s and an atypical G4 structure called a pUG-fold mediate PRC2 dimerization nearly identical to that induced by G4 RNA. In contrast, pyrimidine-rich RNAs, including a motif identified by CLIPseq in cells, do not induce PRC2 dimerization and instead bind PRC2 monomers with retention of methyltransferase activity. Only RNAs that dimerize PRC2 compete with nucleosome binding and inhibit PRC2 methyltransferase activity. CRISPR-dCas9 was adapted to localize different RNA elements onto a PRC2-targeted gene, revealing RNA sequence specificity for PRC2 regulation in cells. Thus, PRC2 binds many different RNAs with similar affinity, however, the functional effect on enzymatic activity depends entirely on the sequence of the bound RNA, a conclusion potentially applicable to any RNA-binding protein with a large transcriptome.

ABSTRACT Evolution simultaneously and combinatorially explores complex genetic changes across perturbation classes, including gene knockouts, knockdowns, overexpression, and the creation of new genes from existing domains. Separate technologies are capable of genetic perturbations at scale in human cells, but these methods are largely mutually incompatible. Here we present CRISPR-All, a unified genetic perturbation language for programming of any major type of genetic perturbation simultaneously, in any combination, at genome scale, in primary human cells. This is enabled by a standardized molecular architecture for each major perturbation class, development of a functional syntax for combining arbitrary numbers of elements across classes, and linkage to unique single cell compatible barcodes. To facilitate use, CRISPR-All converts high level descriptions of desired complex genetic changes into a single DNA sequence that can rewire genomic programs within a cell. Using the CRISPR-All language allowed for head-to-head functional comparisons across perturbation types in a comprehensive analysis of all previously identified genetic enhancements of human CAR-T cells. Combining CRISPR-All programs with single cell RNA sequencing revealed a greater diversity of phenotypic states, including improved functional performance, only accessible through distinct perturbation classes. Finally, CRISPR-All combinatorial genome scale screening of up to four distinct perturbations simultaneously revealed additive functional improvements in human T cells accessible only through iterative multiplexing of modifications across perturbation classes. CRISPR-All enables exploration of a combinatorial genetic perturbation space, which may be impactful for biological and clinical applications.

The constant arms race of bacteriophages and their bacterial hosts has inspired major breakthroughs in biotechnology and shaped phages as fierce predators with great clinical potential to fight multidrug-resistant bacterial pathogens. However, the vast amount of genomic 'dark matter' composed of genes of unknown function in phage genomes remains a major obstacle for the molecular understanding of phage-host interactions. Here we present HIDEN-SEQ, a transposon-insertion sequencing method for phages that systematically links viral genes to selectable phenotypes. Using model phage T4, we show that HIDEN-SEQ readily reproduces the gene essentiality map established over decades of research. Subsequently, we show that our method is easily portable to different phages far beyond classical laboratory models. Across a panel of bacterial hosts and growth conditions, HIDEN-SEQ reveals many conditionally essential phage genes, including previously unknown viral anti-defense factors that we could match to specific antiviral defenses of the respective hosts. Compared to analogous techniques, HIDEN-SEQ provides unprecedented depth and near base-pair resolution as well as great ease of use and portability. We therefore anticipate that HIDEN-SEQ will accelerate discoveries in phage biology by uncovering functions of viral dark matter with direct relevance for microbial ecology, biotechnology, and improvements of phage therapy.

The glycocalyx is best studied for its regulation of immune cell trafficking from the vasculature into inflamed tissues. The glycocalyx is composed of a range of glycans and glyco-proteins that form a peri-cellular matrix and its shedding into the blood is observed in a variety of inflammatory conditions, including viral infections. We now report that macrophages express a glycocalyx that is dependent on their anatomical location and the mediators driving their differentiation from monocytes. Furthermore, during inflammatory conditions caused by viral infection, the macrophage glycocalyx is remodelled in complex ways. Overall, our study provides a novel pathway involved in macrophage inflammatory responses, through remodelling of the glycocalyx halo expressed at steady state, and how this is restored in repair, particularly at mucosal tissue sites.

Abstract When disseminated into the peritoneal cavity at the very early stage, cancer cells must adapt to the glucose- and oxygen-limited environment in peritoneal fluid. To investigate the molecular mechanisms enabling this adaptation, we conducted a genome-wide CRISPR/Cas9 knockout screening in an orthotopic ovarian cancer (OC) model. We identified a series of genes involved in hyaluronic acid (HA) catabolism and glucuronic acid (GlcA) metabolism, including the HA receptor LAYN, HA catabolism enzymes (including HYAL1 and HYAL3) and key GlcA metabolic enzymes (such as AKR1A1 and XYLB). By integrating transcriptomic and metabolic analyses in multiple experimental systems, we demonstrated that HA induced the expression of key HA catabolism and GlcA pathway enzymes, which further led to the release of free GlcA from HA degradation. This GlcA is subsequently metabolized through the GlcA pathway, the pentose phosphate pathway (PPP) and glycolysis to support the maintenance and growth of disseminated OC cells. In addition, we found an atypical Rho GTPase RHOU facilitated the LAYN endosomal recycling for efficient HA uptake. Intriguingly, the rewiring of HA catabolism through GlcA pathway was regulated by its classical receptor CD44 and occurred in other peritoneal disseminating cancers such as bladder cancer and pancreatic adenocarcinoma. Importantly, pharmacological inhibition of HYAL1 with garcinol potently suppressed peritoneal disseminated metastasis in xenograft mice and synergized with cisplatin. In this study, we collectively reported a novel metabolic reprogramming feature of the early peritoneal disseminated cancer cells, which provides new diagnostic and therapeutic strategies for the cancers prone to the potential dissemination.

ABSTRACT Background Parkinson’s disease (PD) involves progressive loss of midbrain dopaminergic (mDA) neurons in the substantia nigra. No disease-modifying treatments exist, only symptomatic relief. Our lab reported an unbiased screen in larval zebrafish identifying renin-angiotensin-aldosterone system (RAAS) inhibitors, including clinically used AGTR1 inhibitors for hypertension, as potent neuroprotective agents. This study aims to investigate the effects of AGTR1 inhibition on human mDA neuron survival using inducible neurodegenerative 2D and 3D models for human mDA neuron degeneration. Methods We report a scalable high-content platform, using CRISPR-engineered human induced pluripotent stem cell (hiPSC)-derived mDA neurons expressing a tyrosine hydroxylase (TH) fluorescent reporter, allowing to track mDA neuron survival live “in a dish”. We developed chemically inducible neurodegenerative 2D and 3D models for human mDA neuron degeneration, allowing to recapitulate PD pathology in human cells in vitro . Results Our model establishes scalable human cellular models of PD well-suited for therapeutic discovery. Using 2D and 3D mono and co-cultures, this study demonstrates that inhibition of AGTR1, via chemical or genetic means, protects against chemically induced mDA neuron degeneration. Transcriptomic analyses show AGTR1 inhibition lowers synuclein transcription, by reducing SNCA and SNCB gene expression. In 3D neuron-glia assembloids, AGTR1 inhibition protects against the accumulation of phosphorylated form of α-synuclein (p129α-Syn), key PD pathological marker. Conclusions We highlight AGTR1 as a key regulator of α-synuclein transcription and aggregation in human mDA neurons, and AGTR1 inhibition as pro-survival in human iPSC-derived models. These findings position inhibition of AGTR1 as a promising therapeutic strategy for PD neuroprotection.

Tumor-infiltrating lymphocyte (TIL) therapy is FDA-approved for patients with treatment-resistant advanced melanoma, but the TIL subpopulations critical for tumor eradication remains incompletely understood. Using patient-derived TIL-melanoma co-cultures, we identified and characterized a novel subset of CD8 + TIL, capable of class I HLA-independent cancer cell lysis. The lymphotoxin β receptor (LTβR) and interferon (IFN) sensing pathways were nominated as key determinants of TIL-mediated cancer cell killing from a whole-genome, loss-of-function CRISPR screen. Validation studies confirmed that dual LTβR and IFN sensing is necessary and sufficient for cancer cell lysis, and that expanded CD8 + TIL express high lymphotoxin β ( LTB ) and upregulate lymphotoxin α ( LTA ) upon coculture with cancer cells. Leveraging paired scRNA-seq and scTCR-seq data, we confirmed that enrichment of LTB + CD8 + T cells is associated with clinical response to TIL, and that LTB + CD8 + TIL are expanded from putative neoantigen-reactive, LTB lo CD8 + T cells in resected tumors. Significance We have uncovered a previously unrecognized mechanism of TIL-mediated tumor eradication, providing mechanistic insights into the role of LTBR/IFN signaling in TIL-mediated cancer cell killing, and potentially offering insights into novel strategies to isolate, enrich, and expand tumoricidal TIL or augment specific TIL functions to enhance tumor control.

Idiopathic short stature (ISS) affects 2%–3% of the population and is genetically heterogeneous, with emerging evidence implicating the extracellular matrix (ECM) of the growth plate. We identify LAMA5 , encoding laminin-α5, as a candidate ISS gene, with rare heterozygous variants present in 1.2% of affected individuals. To define its functional role, we generated CRISPR/Cas9-mediated LAMA5-knockout (KO) urine-derived stem cells (USCs) and induced chondrogenic differentiation in two- and three-dimensional culture systems. Loss of LAMA5 impaired chondrogenesis, with disruption of cell–cell junction programs and abnormal architecture of chondrogenic spheroids. Bulk RNA sequencing combined with weighted gene co-expression network analysis revealed WNT7A and FLI1 as key dysregulated genes within the module most strongly associated with the KO phenotype. Gene Ontology enrichment of this module highlighted embryonic limb morphogenesis as the top biological process, and WNT7A was assigned to canonical WNT signaling. Pharmacologic activation of WNT signaling using lithium chloride (LiCl) partially restored expression of WNT7A , FLI1 , TFAP2A , GRHL2 , and PITX1 toward wild-type levels, indicating that attenuated WNT activity is a principal downstream consequence of LAMA5 deficiency. Consistent with this, we identified an individual with ISS carrying a heterozygous PITX1 missense variant, supporting convergence of ECM (LAMA5) and transcriptional (PITX1) perturbations on a shared WNT-centered limb-morphogenesis network. Together, these findings demonstrate that laminin-α5 is required for proper ECM–WNT signaling integration during human chondrogenesis and suggest that dysregulated WNT activity represents a mechanistic link between LAMA5 dysfunction and impaired endochondral growth. Partial rescue by WNT pathway re-activation highlights a potentially targetable downstream mechanism in ISS pathogenesis.

ABSTRACT CRISPR/Cas9 gene editing revolutionized genetics, but its application is often hampered in non-model plants that are recalcitrant or less amenable to standard plant transformation and regeneration methods. Harnessing viruses to convey guide RNAs (gRNAs) directly to the meristem promises to overcome those limitations and accelerate the generation of edited lines in diverse crops. With several RNA viruses, delivery of gRNAs to the meristem is enhanced with the addition of mobile RNA elements. We hypothesized that incorporating distinct RNA secondary structures as candidate mobility factors in the widely used Tobacco rattle virus (TRV) could propel virus delivery for enhanced meristem editing in non-model species. To test this, we engineered TRV vectors to deliver gRNAs targeting visible marker genes, with each virus incorporating unique mobility factors. We determined optimal virus construction for multiplexed meristem editing by first delivering each virus to Nicotiana benthamiana plants harboring the Cas9 transgene. Strikingly different phenotypes were observed among virus treatments, which were confirmed to represent distinct somatic and heritable editing events. We further tested our hypothesis by leveraging these results to edit pennycress ( Thlaspi arvense ), an emerging oilseed crop. Our results demonstrated successful virus delivery of meristem editing to this non-model plant, underscoring the potential of this approach to deliver targeted genome modifications in diverse crops. One sentence summary Incorporating RNA mobility factors in TRV affects meristem editing in model plants and crops.

Presence of pathogenic viruses in wastewater pose a potential threat to public health. Conventional treatment methods often yield moderate viral reduction and toxic byproducts, whereas advanced technologies are underutilised due to their high cost and energy demands. Antiviral phytoremediation emerges as an affordable, eco-friendly and sustainable approach for removing viruses. However, recent bibliometric analysis on wastewater treatment methods from 1976–2025 revealed that only ~0.4% of total literature (~23,000) was related to antiviral phytoremediation suggesting critical knowledge gaps persist. This critical review provides insights into viral removal mechanisms, recent advancements, practical applications, and challenges and opportunities. Antiviral phytoremediation offers a promising multilayer of viral removal mechanisms (i.e., sorption/filtration, rhizosphere-mediated inactivation, internalization, and intracellular degradation mechanisms). Hybrid systems integrating constructed wetlands (CWs) with complementary technologies could achieve high removal efficiencies (i.e., ∼3.0–7 log₁₀ reductions) compared to standalone CWs (i.e., ∼1–3 log₁₀). Although phytoremediation efficiency is moderate for viruses (i.e., ∼45–84%) relative to heavy metal removal (i.e., ∼70–100%), emerging technologies (i.e., CRISPR gene editing, engineered microconsortia, and biosensors) offer promise for enhancement, which is still at proof-of-concept levels. Hybrid antiviral phytoremediation approaches provide sustainable infrastructure supporting public health, climate adaptation, and pandemic preparedness.

The first lineage decision in the mammalian blastocyst commits outer cells to the trophectoderm and initiates the trajectory that gives rise to the placental chorion. The molecular sequence that unfolds downstream of HIPPO pathway inactivation, linking human trophectoderm specification to the early organization of the chorion, has remained unknown. Here, we establish a developmentally informed model that leverages HIPPO pathway modulation to induce the native trophectoderm trajectory in the absence of exogenous BMP or WNT signaling. We first transiently reset primed human pluripotent stem cells into a trophectoderm-competent ground state, followed by LATS kinase inhibition to set the trajectory in motion. To benchmark fidelity, we built an embryo-chorion single-cell reference integrating published early human and placental transcriptomes and applied a computational stage-matching tool to align our cultures to natural development. Stage matching revealed an ordered progression along the trophectoderm trajectory from early TE to post-implantation trophoblast. With extended culture, all major cell types of the nascent chorion emerged, encompassing both trophoblast and chorionic mesoderm lineages. Within the trophoblast, we identified proliferative and non-cycling villous cytotrophoblast, a columnar population connecting villous and extravillous domains, as well as syncytia and extravillous subtypes. When cultured in suspension, these lineages self-organized into three-dimensional organoids that recapitulated the stromal-epithelial architecture and proliferative-syncytial polarity of the emergent chorion. We identified CLDN6 as a defining surface marker of columnar trophoblast, the population that bridges villous and extravillous compartments. Prospective isolation of living CLDN6+ trophoblast revealed their capacity to reacquire a proliferative villous state and, under directed cues, generate both syncytial and extravillous fates, confirming their proposed dual developmental potential within the chorion. Together, these findings establish a developmentally informed framework that connects human trophectoderm specification to the emergent chorion and provides a dynamic platform for investigating the earliest steps of placental specification and the origins of implantation disorders.

ABSTRACT Predicting how changes in human DNA sequence impact gene expression remains challenging. Here, we present PETRA ( P rime E diting of T ranscribed R egulatory elements to A ssay expression), a multiplexed genome editing method to quantify the effects of regulatory variants at scale. PETRA leverages the delivery of variants to abundantly transcribed regions of genes such that sequence-specific effects on mRNA expression can be read out by amplicon sequencing. We demonstrate PETRA in Jurkat cells by scoring 13,935 six-nucleotide insertions delivered to the 5’ untranslated regions (5’ UTRs) of four genes important for T cell responses, namely VAV1 , IL2RA , CD28 and OTUD7B . Effects on expression are linked to the creation of new transcription factor binding sites (TFBSs), as well as to alterations in splicing and translation initiation. Combinatorial delivery of TFBSs identified using PETRA enables the discovery of alleles that increase mRNA expression more than 10-fold. Lastly, we extend PETRA to primary human T cells and compare variant effects across cell types. These experiments establish PETRA as a flexible means of dissecting the logic of gene regulation across genomic contexts and cell types.

Cancer immunotherapy has recently become an essential approach for treating cancer, showing considerable promise as a substitute for surgery, radiation therapy, and conventional chemotherapy. It primarily aims to boost the host’s natural defense system to com-bat cancer malignancies by utilizing components of immune checkpoint blockades (ICBs), mainly programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), and along with elements of adoptive cellular therapies (ACTs) like Chimeric Antigen Receptor (CAR) therapy, T Cell Receptor (TCR) therapy and Tu-mor-Infiltrating Lymphocyte (TIL) therapy. However, cancer cells tend to undermine the effectiveness of cancer immunotherapeutic strategies by employing one or more immune evasion mechanisms. The present review briefly discusses the key mechanisms of cancer immune evasion and highlights how the CRISPR/Cas9 systems, as gene editing tools, are set to enhance cancer immunotherapy for treating various challenging cancers. We emphasize that CRISPR/Cas9 systems can be used to explore and positively alter the genes of the immune system, boosting the effectiveness of cancer immunotherapy by editing immune checkpoints, TILs, and CAR-T cells, and disrupting genes facilitating tumors to evade the immune system.

Neurodevelopmental disorders (NDDs), including autism spectrum disorder (ASD), attention-deficit/hyperactivity disorder (ADHD) and intellectual disability (ID), arise from disruptions of tightly orchestrated molecular programmes that govern neurogenesis, synaptogenesis and circuit maturation. Although large-scale genomic analyses have identified numerous susceptibility loci, DNA variation alone explains only a fraction of disease heritability, highlighting the pivotal contribution of post-transcriptional and epigenetic regulation. Among these regulatory layers, non-coding RNAs (ncRNAs)—encompassing microRNAs (miRNAs), long non-coding RNAs (lncRNAs), circular RNAs (circRNAs), PIWI-interacting RNAs (piRNAs) and transfer-RNA-derived small RNAs (tsRNAs)—have emerged as key modulators of neural differentiation, synaptic plasticity and intercellular communication. Multi-omics studies reveal that ncRNAs fine-tune chromatin accessibility, transcriptional output and translation through complex competing-endogenous-RNA (ceRNA) and ribonucleoprotein networks. While miRNAs sculpt neurogenesis and circuit remodelling, lncRNAs and circRNAs integrate chromatin and transcriptional control with exquisite spatial and temporal precision. Newly characterized small RNAs such as tsRNAs and piRNAs extend this regulatory repertoire by linking translational reprogramming, epigenetic memory and even intergenerational inheritance. Advances in single-cell and spatial transcriptomics have further mapped ncRNA expression to discrete neuronal and glial populations, revealing cell-type-specific vulnerability signatures across cortical and subcortical regions. Clinically, circulating ncRNAs—particularly those encapsulated within plasma or extracellular vesicles—exhibit robust and disease-specific expression patterns, supporting their promise as non-invasive biomarkers for early diagnosis and patient stratification. In parallel, innovations in RNA interference, antisense oligonucleotides, CRISPR-based editing and exosome-mediated delivery are transforming ncRNAs from molecular indicators into therapeutic instruments capable of restoring transcriptional and epigenetic equilibrium. Together, these converging insights position ncRNAs as both mechanistic determinants and translational targets in neurodevelopmental pathology. The emerging ncRNA landscape redefines the molecular architecture of brain development, offering a unifying framework that links genome regulation, environmental responsiveness and neural plasticity. Decoding this multilayered RNA circuitry will be pivotal for the development of next-generation diagnostics and RNA-guided therapies for neurodevelopmental disorders.

The rice genome encodes five non-expressors of pathogenesis-related (NPR) homologs, with OsNPR1/NH1 and OsNPR3/NH3 emerging as pivotal players in salicylic acid (SA)-mediated defense responses. Investigating the functional implications of the remaining NPR/NH genes is critical for the development of disease-resistant rice cultivars. This study explores the role of OsNH2 in rice defense against sheath blight (ShB) using CRISPR/Cas9-edited mutants of the susceptible cultivar ASD16 and the moderately resistant CO51. OsNH2 knockout mutants showed increased susceptibility to ShB, as evidenced by dense mycelial growth, wider hyphae, and elevated superoxide radical content. Two in-frame deletion mutants lacking 15–17 amino acids in the BTB/POZ domain also showed higher susceptibility, highlighting the importance of an intact OsNH2 protein for resistance. qRT-PCR analysis revealed significant downregulation of OsNH1 , OsNH3 , key transcription factors ( WRKY4 , WRKY45 , WRKY80 , TGA2 and TGA3 ), pathogenesis-related (PR) genes ( PR1 , PR3 and PR5 ), and SA biosynthesis genes ( PAL and ICS1 ) in the mutants. Additionally, OsNH2 mutants in both cultivars exhibited reduced endogenous SA levels upon Rhizoctonia solani infection. Exogenous SA treatment partially restored resistance and upregulated OsNH1/3 expression in mutants, though not to wild-type levels. These results suggest that OsNH2 is essential for maintaining SA-mediated defense signaling and optimal expression of NPR1 homologs. Moreover, OsNH2 mutants also showed increased susceptibility to bacterial leaf blight (BLB). Collectively, this research highlights the critical role of OsNH2 in coordinating with OsNH1 and OsNH3 in SA-mediated defense against ShB and BLB in rice. Highlights CRISPR/Cas9-edited OsNH2 knockout mutants, along with in-frame deletion mutants lacking 15–17 amino acids in the BTB/POZ domain, exhibited increased susceptibility to sheath blight disease in rice. OsNH2 disruption led to reduced endogenous salicylic acid (SA) levels and significant downregulation of OsNH1 , OsNH3 , key WRKY and TGA transcription factors, and pathogenesis-related (PR) genes. Exogenous SA treatment partially restored resistance and upregulated OsNH1 / 3 expression in mutants, though not to wild-type levels—highlighting OsNH2’s essential role in sustaining SA-mediated defense signaling. OsNH2 mutants also showed increased susceptibility to bacterial leaf blight (BLB), emphasizing its coordination with OsNH1 and OsNH3 in defense against multiple rice pathogens. Graphical abstract

Genome integrity is challenged by DNA damage. DNA double-strand breaks are the most harmful DNA lesions as they block DNA replication and transcription leading to chromosome reorganisations or cell death if not properly repaired. In addition, increasing evidence points to chromatin as a relevant modulator of the efficiency of repair in eukaryotes. Here, we show that inhibition or depletion of human histone deacetylase 1 (HDAC1) regulates DSB repair by controlling the phosphorylation of H2AX, an early step of the DNA damage response. Thus, DSB repair is regulated by a crosstalk between histone acetylation and phosphorylation. Our study provides evidence that histone acetylation regulates DSB signalling supporting that histone deacetylase inhibitors could enhance genotoxic treatment of cancer.

ABSTRACT Biallelic AAGGG expansions in Replication Factor Complex Subunit 1 ( RFC1) are associated with cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS) and are increasingly recognised as a common cause of adult-onset ataxia and sensory neuropathy. However, the disease-causing mechanisms remain unclear. Here we leveraged in vitro assays, post-mortem brain tissue, patient-derived cell lines and a neuronal Drosophila model to demonstrate that AAGGG expansions are associated with tissue-specific reductions in the expression of RFC1 transcript, along with impaired RFC1 function and increased sensitivity to DNA damage from platinum-based drugs. CRISPR/Cas9 excision of the AAGGG repeat and flanking AluSx3 element normalized RFC1 expression in iPSC-derived neurons and rescued the DNA damage response, providing a framework for future therapeutic strategies. We also show that these biological findings are clinically relevant in heterozygous AAGGG expansion carriers, who display an increased risk and severity of neuropathy with platinum-based chemotherapy.

ABSTRACT Despite their well-defined genetic background, repeat expansion diseases (REDs) still represent an unmet medical need, with no causative therapy offered to patients. The strategy of repeat shortening using genome editing tools is very attractive because a single intervention can result in permanent repair of the disease-causing mutation. However, a limited understanding of DNA repair mechanisms in repetitive sequences complicates the prediction and control of the editing effects. Using a CRISPR interference (CRISPRi) screen, we identified pathways and factors responsible for the repair of staggered cuts generated by Cas12a within CAG repeat tracts. This analysis revealed a central role for interstrand crosslink (ICL) repair factors in mediating CAG repeat contraction, with DCLRE1A emerging as a key effector. We demonstrated that DCLRE1A recognizes and binds structures generated by Cas12a. Moreover, DCLRE1A interacts with SLX4, promoting the generation of pure contractions, and with POLI, leading to the formation of inverted repeats at the break site during template switching mechanisms. We then used this knowledge to increase the contribution of pure contractions to the pool of editing outcomes using fusions of Cas12a with DNA repair proteins. Our study indicates that Cas12a can be used as an effective tool for generating repeat contractions. Although the mechanisms leading to repeat shortening are complex, understanding them can help researchers develop more precise therapeutic strategies with greater control of the editing process.

IFIT1 is among the highest expressed proteins following virus detection and interferon (IFN) induction and binds the 5’-end of ‘non-self’ viral cap0-mRNAs lacking 2’-O-methylation, blocking their translation. IFIT1 hetero-complexes with IFIT3, which enhances IFIT1 cap0-binding. Here, we demonstrate that IFIT3 is a master regulator of IFIT1, dictating its activity and stability. When expressed at high levels in the absence of IFIT3, IFIT1 can inhibit Semliki Forest virus replication, but can also inhibit translation of certain other ‘self’ IFN-stimulated genes (ISGs), including the important innate immune proteins ISG15 and IFITM1 as exemplars. However, IFIT1:IFIT3 complexing rescues ISG15 and IFITM1 from IFIT1 translation inhibition. We demonstrate that IFIT1 is degraded by the proteasome in the absence of IFIT3, but that direct binding to IFIT3 protects IFIT1 from degradation, ensuring IFIT1 accumulation only occurs along with IFIT3, demonstrating a mechanism to control the fine balance between antiviral activity and self-targeting in the face of unrelenting viral evolution.

Breast cancer recurrence remains a major clinical challenge, often associated with therapy resistance and altered metabolic states. To define metabolic vulnerabilities of recurrent disease, we performed a CRISPR knockout screen targeting 421 metabolic genes in paired primary and recurrent HER2-driven breast cancer cell lines. While both primary and recurrent tumors shared dependencies on core metabolic pathways, recurrent tumors exhibited selective essentiality for the de novo pyrimidine synthesis pathway, including Cad , Dhodh , and Ctps . Pharmacologic inhibition of the rate-limiting enzyme DHODH with BAY-2402234 selectively impaired the growth of recurrent tumor cells, while primary tumor cells were relatively resistant. BAY treatment robustly inhibited pyrimidine synthesis in all lines, but only recurrent cells underwent iron-dependent lipid peroxidation and ferroptotic cell death. Lipidomic profiling revealed enrichment of polyunsaturated ether phospholipids in recurrent cells, which may predispose them to ferroptosis. A sensitizer CRISPR screen in primary cells further identified nucleotide salvage and lipid metabolic pathways as modifiers of DHODH inhibitor sensitivity. Stable isotope tracing and nutrient depletion experiments showed that primary cells can compensate for DHODH inhibition through nucleotide salvage, whereas recurrent cells exhibit impaired salvage capacity, likely due to reduced expression of Slc28 / Slc29 nucleoside transporters. Together, these findings reveal that breast cancer recurrence is associated with increased dependence on de novo pyrimidine synthesis to suppress ferroptosis, highlighting a therapeutically actionable metabolic vulnerability in recurrent disease.

Abstract SARS-CoV-2 is a positive-sense RNA virus and was responsible for the devastating COVID-19 pandemic. Although the current disease burden is less severe, there are limited treatment options, significant gaps in knowledge, and a looming threat of the emergence of variants and future pandemics. To address these challenges, we performed genome-wide CRISPR knockout screens in a novel human lung cell line NCI-H23 ACE2 , as well as in HEK293T ACE2 cells, with SARS-CoV-2 Wuhan virus, with the aim of identifying host-dependency factors that could predict effective antivirals. We identified four host-directed drugs, donepezil, dH-ergocristine, trametinib and sorafenib, that could potentially be repurposed to treat coronavirus infections. Three of the drugs inhibited SARS-CoV-2, HCoV-229E, and HCoV-OC43, suggesting they could be used as pan-coronavirus antivirals. We also confirmed that SARS-CoV-2 relies on the NRAS/Raf/MEK/ERK signaling pathway for its replication. Our study highlights the robustness and efficiency of a bilateral approach of gene silencing and antiviral screening to identify host-dependency factors and effective antivirals.

The advent of CRISPR-based genome editing has transformed the conceptual framework of oncology—from descriptive molecular profiling to functional genome engineering. By enabling precise, programmable, and multiplex control of cancer-associated genes, CRISPR/Cas systems are reshaping how we model tumorigenesis, predict drug response, and design patient-tailored interventions. This Perspective discusses how CRISPR technologies are redefining precision oncology, the biological and ethical challenges that impede their clinical translation, and emerging strategies that integrate gene editing with immunotherapy, synthetic biology, and systems medicine. We argue that the future of cancer therapy lies not merely in editing genes but in orchestrating the dynamic networks that sustain malignancy.