CRISPR-Cas13d is increasingly used for RNA knockdowns due to its programmability, but off-target RNA binding and cleavage of near-cognate RNAs hinder its broader adoption. Here, we explore the mechanisms of nuclease activation by solving seven ternary cryo-electron mi-croscopy structures of wild-type Cas13d in complex with matched and mismatched targets. These structures reveal a series of active, intermediate, and inactive states that illustrate a detailed activation mechanism. The crRNA undergoes dramatic conformational changes upon target RNA binding, with the helical-1 domain transitioning from an initially docked state with the N-terminal domain to an allosterically switched conformation that stabilizes the RNA duplex. Quantitative kinetics reveal that a single proximal mismatch preserves nanomolar binding affinity but completely abolishes nuclease activity by trapping Cas13d in an inactive state. We identify an active site loop in the HEPN domains that regulates substrate accessibility, with alanine scanning mutagenesis revealing both hypo- and hyperactivated variants. These findings establish the structural basis for Cas13d’s exquisite mismatch surveillance and provide a mechanistic framework for engineering RNA-targeting specificity and activity across HEPN nuclease family members.

The balance between excitatory and inhibitory neurotransmission is fundamental for normal brain function, yet the adaptation of individual neurons to disrupted excitatory-inhibitory balance is not well understood. We developed highly efficient, in vivo RNA electroporation-based single-cell gene editing to investigate neuronal responses to loss of fast inhibition. Using CRISPR-Cas9 components delivered as RNA, we knocked out GABA-A receptor β subunits in individual layer 2/3 cortical neurons in mouse visual cortex, eliminating fast inhibition. In vivo patch-clamp recordings revealed that cortical neurons adapted to inhibition loss through two sequential mechanisms: a transient reduction of excitatory synaptic input, followed by intrinsic membrane property changes that decreased input resistance. This sequential adaptation program ultimately prevented target neurons from contributing spikes to the cortical network. Our RNA-based single-cell gene editing approach enables investigation of cellular responses independent of network effects, providing new insights into neuronal homeostasis and gene function in individual cells in vivo.

Abstract Background: The plant circadian clock is crucial for regulating developmental and metabolic processes, enabling crops to adapt to environmental changes and maintain high productivity. In rice, the clock gene OsPRR37 plays a pivotal role in photoperiod sensitivity and the regulation of yield-related traits. However, the complete regulatory network of OsPRR37 remains largely unexplored. Results: This study utilized an integrated multi-omics approach, combining transcriptome profiling, DNA affinity purification sequencing (DAP-seq), and protein–protein interaction (PPI) mapping to construct a multi-layered regulatory model of OsPRR37 . CRISPR/Cas9 knockout lines showed significant changes in flowering time, plant height, panicle architecture, and spikelet number. Transcriptome analysis associated OsPRR37 with pathways related to photosynthesis, carbohydrate metabolism, and stress responses. Comparative analysis of knockout and overexpression datasets identified 454 candidate target genes exhibiting inverse expression patterns, including regulators of flowering and chlorophyll biosynthesis. DAP-seq revealed 1,679 high-confidence DNA-binding sites, with nine genes identified as direct targets, six of which contained conserved motifs associated with cytokinin signaling, inflorescence architecture, and meristem determinacy. PPI mapping through a yeast two-hybrid screen identified 26 interacting proteins, including OsGlyRS3 and OsSnRK1A, which are involved in flowering, sugar signaling, chloroplast development, and hormone metabolism. Structural modeling suggested that OsGlyRS3 may stabilize OsPRR37 protein complexes, while OsSnRK1A could modulate its DNA-binding capacity under sugar-deficient conditions. Conclusions: The findings establish OsPRR37 as a central regulatory hub that coordinates flowering, energy metabolism, chloroplast function, and stress adaptation through a hierarchical network comprising a Modulatory Layer of protein interactors, a Direct Target Layer of DNA-bound genes, an Indirect Coherent Layer of transcriptional cascades, and a Diffuse Response Layer encompassing broad metabolic outputs. This model provides a comprehensive framework for understanding how OsPRR37 integrates circadian signals to control multiple agronomic traits and offers valuable targets for breeding climate-resilient, high-yielding rice varieties.

Abstract Triple-negative breast cancer (TNBC) is a subtype with limited treatment options and poor outcomes, particularly in the metastatic setting. Although immunotherapy has shown efficacy in early-stage disease, its benefit remains suboptimal in women with locally advanced and metastatic TNBC. Here, we identify the splicing factor PTBP1 as a tumor-intrinsic regulator of immune evasion in metastatic TNBC. By integrating clinical, single-cell, and bulk transcriptomic data with multiplex immunohistochemistry, CRISPR-Cas9 genome editing, and functional assays, we show that PTBP1 impairs antigen presentation, promotes T cell dysfunction, and is associated with worse outcomes, independent of tumor-infiltrating lymphocyte levels. Furthermore, CRISPR-mediated silencing of PTBP1 restores HLA expression and reactivates antigen presentation pathways in TNBC. PTBP1 expression is elevated in metastatic compared to primary TNBC tumors and correlates with immune dysfunction signatures. Consistently, in the phase II TONIC clinical trial, metastatic TNBC patients with PTBP1-high tumors had poor response and shorter survival following PD-1 blockade, and PTBP1 expression showed a predictive performance comparable to PD-L1 and TILs in this cohort. These findings position PTBP1 as a tumor-intrinsic regulator of immune evasion and a potential biomarker to inform immunotherapy strategies in metastatic TNBC.

Abstract Background RFX3 is a transcription factor (TF) critical for pancreatic endocrine development. Its loss impairs β-cell differentiation, promotes enterochromaffin cell (EC) generation, and induces apoptosis. However, the role of non-coding RNAs in mediating these effects remains poorly understood. Methods Using CRISPR/Cas9-derived RFX3 knockout (KO) human iPSC-derived pancreatic islets, we performed integrated transcriptomic analyses of coding and non-coding RNAs. Differentially expressed miRNAs (DEmiRs) and lncRNAs (DElncRNAs) were validated by RT-qPCR. Target prediction and competing endogenous RNA (ceRNA) network analyses were conducted to explore potential regulatory interactions affecting key endocrine genes. Results RFX3 deficiency induced widespread transcriptomic changes in human iPSC-derived pancreatic islets. Core β-cell markers and key pancreatic TFs were downregulated, alongside genes regulating ion channels, vesicle trafficking, metabolic sensing, and insulin secretion. Conversely, apoptotic and EC genes were upregulated. RFX3 KO islets exhibited significant alterations in miRNA profiles, including upregulation of miR-451a, miR-215-5p, miR-122-5p, miR-338-3p, miR-194-5p, miR-378a-3p , and the miR-29 family, which are predicted to target critical pancreatic endocrine genes. Several lncRNAs, including LINC00461, MIAT, RMST , and AC020916.1 , were downregulated, potentially influencing miRNA activity via ceRNA interactions. Integrated analyses identified core regulatory axes, such as miR-4455/INS, miR-122-5p/ARX, and miR-660-3p/GHRL, while apoptosis-related genes, including CASP, TNFSF10 , and TXNIP , were also predicted targets of dysregulated miRNAs. Conclusions Our findings reveal widespread alterations in non-coding RNA networks associated with RFX3 loss, highlighting a potential layer of post-transcriptional regulation linked to impaired pancreatic endocrine development. These results provide insights into how RFX3 deficiency may reshape islet transcriptomes, influencing β-cell maturation, function, and survival.

Leptospirosis is a globally distributed zoonotic disease caused by pathogenic bacteria of the Leptospira genus. Genome editing in Leptospira has been difficult to perform. Currently, the functionality of the CRISPR-Cas system has been demonstrated in species such as Leptospira interrogans. However, the different CRISPR-Cas systems present in most of the 77 species are unknown. Therefore, the objective of this study was to identify the CRISPR-Cas systems present in the genomes of the Leptospira genus using bioinformatics tools. Methods: bioinformatics workflow was followed: the genomes were downloaded from the NCBI database, Cas proteins detection was carried out using the CRISPR-CasFinder and RAST web servers, functional analysis of Cas proteins (InterProScan, ProtParam, Swiss Model, Alphafold3, Swiss PDB Viewer, and Pymol), conservation pattern detection (MEGA12, and Seqlogos), spacer identification (Actinobacteriophages db and BLAST), and bacteriophage detection (Phaster, and Phastest). Results: Cas proteins were detected in 36/77 species of the Leptospira genus, these proteins were (Cas1-Cas9, and Cas12). The proteins were classified into class 1 and class 2 systems, and types I, II, and V. Direct repeats and spacers were detected in 19 species. The direct repeats presented two nucleotide conservation motifs. With the spacer sequences, 270 different bacteriophages were identified. Three intact bacteriophages were detected in the genome of four Leptospira species. Two saprophytic species have complete CRISPR-Cas systems. Conclusions: The presence of Cas proteins, direct repeats, and spacer sequences homologous to bacteriophage genomes suggests a functional CRISPR-Cas system in at least 19 species.

Mating in insects commonly induces an alteration in behavior and physiology in the female that ensures optimal offspring. This is referred to as a post-mating response (PMR). The induction of a PMR requires not only male-derived factors transferred with semen during copulation, such as sex peptide (SP) in Drosophila , but also intrinsic female signaling components. The latter signaling remains poorly understood in most insects, including the brown planthopper (BPH) Nilaparvata lugens , a devastating rice pest. In BPHs the PMR comprises a reduced receptivity to re-mating and increased oviposition. Here, we demonstrate that the neuropeptide corazonin (CRZ) and its receptor (CrzR) are critical for the PMR in female BPHs. Peptide injection and knockdown of CRZ expression by RNAi or CRISPR/Cas9-mediated mutagenesis demonstrate that distensible CRZ signaling suppresses mating receptivity in virgin N. lugens females and mediates a reduction in re-mating frequency and increased ovulation. The CrzR is highly expressed in the female reproductive tract, and CrzR -knockdown phenocopies Crz diminishment. Importantly, female CRZ/CrzR signaling is indispensable for male seminal fluid factors (e.g. maccessin) to induce the PMR. With disrupted CrzR signaling, seminal fluid or maccessin injection fails to reduce female receptivity. Notably, CRZ is not produced in male accessory gland (MAG) and thus not transferred during copulation. However, male Crz knockout impairs the PMR in mated females and combining male and female Crz knockouts nearly abolished the PMR, demonstrating that CRZ is essential for PMR generation. Transcriptomics of the MAG indicates that Crz knockout affects the expression of numerous seminal fluid protein genes. Finally, we found that also in female Drosophila melanogaster , disrupted Crz signaling resulted in increased re-mating and reduced oviposition, while CRZ injection suppressed virgin receptivity and increased oviposition. In summary, our study reveals that endogenous female CRZ signaling and male-derived signals cooperate to regulate post-mating transitions in BPHs and Drosophila .

Wheat ( Triticum aestivum L.) is one of the most important crops worldwide, supplying a major share of calories and protein for the global population. Incorporating gene editing into breeding programs is critical to improve yield and stress tolerance, yet wheat remains difficult to transform and regenerate efficiently. These bottlenecks limit the full application of CRISPR/Cas9 for improvement yield in wheat. To address this, transformation parameters were optimized for three methods: immature embryo transformation, callus transformation, and injection-based in planta transformation. Systematic optimization of Agrobacterium strain, bacterial density, acetosyringone concentration, and incubation conditions resulted in substantially improved transformation success. Efficiencies of 66.84% for immature embryos, 55.44% for callus, and 33.33% for in planta transformation were achieved, representing more than tenfold increase compared with previously reported rate of ∼3%. A key innovation was the shortening of the callus induction stage for immature embryos, reducing the time required for plant regeneration by approximately one month while maintaining high transformation efficiency. The protocols were validated through CRISPR/Cas9-mediated knockout of TaARE1-D , a negative regulator of nitrogen uptake and yield. Generated mutants exhibited increased grain number, spike length, grain length, and thousand-grain weight, as well as the characteristic stay-green phenotype associated with loss of TaARE1-D function. The optimized protocols provide robust platforms to accelerate gene-editing in wheat to increase yield and stress-tolerance.

Protein kinases are key regulators of the eukaryotic cell cycle and have consequently emerged as attractive targets for drug development. Their well-defined active sites make them particularly amenable to inhibition by small molecules, underscoring their druggability. The Leishmania kinome, shaped by diverse evolutionary processes, harbours a unique repertoire of potential drug targets. Here, we used the cysteine-directed protein kinase probe SM1-71 to identify four essential protein kinases MPK4, MPK5, MPK7 and AEK1 as candidates for covalent kinase inhibitor development, as well as CLK1/CLK2 for which covalent inhibitors have already been identified. We leveraged the absence of natural analog-sensitive (AS) kinases in L. mexicana to establish an in vivo chemical-genetic AS kinase platform for investigating essential functions of protein kinases. Using CRISPR-Cas9-mediated precision genome editing, we endogenously engineered two kinetochore-associated protein kinases, KKT2 and KKT3, and cyclin-dependent kinase CRK9, to generate AS kinases. We show that KKT2 and CRK9 kinase activities are essential for both promastigote and intracellular amastigote survival; KKT2 kinase activity being required for progression through mitosis at a stage preceding mitotic spindle assembly, while CRK9 kinase activity is required for S phase, consistent with its role in trans-splicing. This study demonstrates the utility of AS chemical genetics in Leishmania and identifies KKT2 and CRK9 as having critical roles in Leishmania cell cycle regulation and therefore being promising drug targets.

Fatty acid desaturase 12 (FAD12) is a key enzyme in fatty acid biosynthesis, responsible for converting oleic acid to linoleic acid through desaturase activity. Euglena gracilis (Euglena) is an emerging platform for the industrial production of various metabolites, including lipids. However, a comprehensive understanding of Euglena’s fatty acid biosynthesis pathways remains incomplete, posing a significant barrier to the commercialization of Euglena bioproducts. To address this gap, we employed a bioinformatics approach to identify a Euglena gracilis FAD12 ( Eg FAD12). We analyzed the evolutionary relationship of Eg FAD12 with its homologs from other organisms and revealed that the three canonical histidine box motifs are conserved among FAD12s. To characterize Eg FAD12, we cloned it into the pEAQ-hyperstrans vector and overexpressed it in Nicotiana benthamiana to take advantage of its endogenous fatty acid pool, which could act as substrates. The heterologous expression of FAD12 in N. benthamiana led to an increased linoleic acid content, demonstrating the suspected desaturase activity. To further confirm the function of Eg FAD12, we performed CRISPR-Cas9-mediated knockout of Eg FAD12 in Euglena, which resulted in a drastic reduction in linoleic acid (C18:2) without compromising biomass yield or lipid content. This work advances our understanding of fatty acid biosynthesis in Euglena and will aid in its adoption as a platform for producing customized lipids.

Widespread biodiversity loss driven by human activity has intensified global efforts to restore degraded ecosystems. Yet a key challenge remains: how to track restored individuals and their offspring over time and space to assess the true impact of restoration? This is especially pressing for coral reefs, which support a quarter of all marine species, are in severe decline globally, and are now the focus of growing restoration initiatives worldwide. Here, we demonstrate a novel approach that combines genome editing and environmental DNA (eDNA) monitoring to enable scalable, non-invasive tracking of individual corals and their offspring. Using CRISPR-Cas9, we introduced unique genetic barcodes into non-coding regions of the Acropora millepora genome. These barcodes - stable, heritable, and distributed across the genome - will allow for individual-level identification over multiple generations. We show that these barcodes can be reliably detected in surrounding seawater using eDNA metabarcoding, offering a powerful, non-destructive tool for tracking corals in situ . Together, this approach provides a proof-of-concept for precision monitoring of restoration outcomes, with broad applicability across species and ecosystems.

Bone morphogenetic proteins (BMP) induce apoptosis in myeloma cells and the mechanism behind this could point to new therapeutic targets. Here, we did a whole genome CRISPR/Cas9 knockout screen using the INA-6 myeloma cell line. Apoptosis was induced with BMP9 and the relative amounts of sgRNAs in treated versus control cells were determined with next-generation sequencing. We identified key positive control genes and a substantial number of novel genes that could be involved in BMP-induced apoptosis. One of the overrepresented genes was the known BMP target gene ID3 . We found that ID3 was potently induced by BMP9 treatment and that depletion of ID3 protected cells from c-MYC downregulation and apoptosis. ID3 is known to heterodimerize with basic helix-loop-helix (bHLH) TCF transcription factors. In the screen, TCF3 , TCF4 , and TCF12 were among genes that potentially protected cells from apoptosis. Knockdown of TCF3 , and to some extent TCF12 , led to lower basal c-MYC levels and lower cell viability, and this was more pronounced after BMP9-treatment. Our results suggest that ID3 plays an important role in regulating the survival of myeloma cells, at least in part by forming heterodimers with TCF3 and thus preventing expression of the c-MYC oncogene.

Summary Parvovirus B19 (B19V) is a prevalent human pathogen that can cross the placenta by a mechanism that remains unknown, posing a risk of severe fetal complications, particularly during the first trimester of pregnancy. We investigated the expression of B19V-specific receptors in the three trophoblast cell types, cytotrophoblasts (CTBs), syncytiotrophoblasts (STBs), and extravillous trophoblasts (EVTs), and assessed their susceptibility to infection. VP1uR, the erythroid-specific receptor that mediates viral uptake and infection in erythroid progenitor cells, is expressed in CTBs and STBs, but not in EVTs. Globoside, a glycosphingolipid that is essential for the escape of the virus from endosomes, is also expressed in these cells, except for choriocarcinoma-derived CTBs. In the latter, the absence of globoside can be overcome by promoting endosomal leakage with polyethyleneimine. While erythropoietin receptor (EpoR) signaling is associated with the strict erythroid tropism of B19V, it is not required for infection in trophoblasts. Transfection experiments revealed that highly proliferative first-trimester CTBs are more permissive to B19V infection than the low-proliferative CTBs from term placenta. These findings demonstrate that B19V targets and infects specific trophoblast cells, where viral entry and replication are collectively mediated by VP1uR, globoside, and high cellular proliferative activity, but are independent of EpoR signaling. Highlights Trophoblasts express the specific receptors necessary for B19V entry. Susceptibility and permissiveness to B19V vary by trophoblast subtype and gestational age. Highly proliferative first-trimester cytotrophoblasts show increased permissiveness to B19V. B19V infection in trophoblasts depends on globoside but is independent of EpoR signaling.

Chronic widespread pain (CWP) is a complex condition linked to impaired arterial health, including atherosclerosis and increased arterial stiffness. Epidemiological evidence suggests shared biological mechanisms, with strong associations between CWP and arterial dysfunction, However, the genetic basis remains largely unexplored. We conducted a common pathway genome-wide association study using genomic structural equation modeling (GenomicSEM) and GWAS summary statistics for CWP, atherosclerosis, and pulse wave velocity to identify shared genetic factors. This analysis revealed 53 genome-wide significant variants contributing to a shared latent factor, with opposing trait loadings suggestive of antagonistic pleiotropy. Lead loci included RNF123, ATP2C1, and COMT, with gene-level analysis implicating neurodevelopmental pathways and glycosaminoglycan degradation through hyaluronidase activity. Chromatin interaction and expression mapping supported regulatory links in relevant tissues. Our findings demonstrate that neurogenic and extracellular matrix-related processes, including glycan metabolism, contribute to the shared genetic architecture of CWP and cardiovascular traits, offering mechanistic insight into their comorbidity.

Most of the environmental flavobacteria decompose organic matter, playing a crucial role in ecosystem balance. Phages infecting these bacteria regulate the host populations and thereby the ecosystem functions, however, bacterial resistance against phage may cause changes in bacterial phenotypic characteristics. ssDNA phage Finnlakevirus FLiP infects three known environmental Flavobacterium sp. isolates: B330, B167, and B114. Building on our previous FLiP-host interaction studies, we aimed to broaden our understanding of the host perspective by exploring phage resistance mechanisms against FLiP and similar phages. We compared growth dynamics of ancestral and resistant variants to detect the effect of resistance on host fitness. Genomic comparison between the variants was used to identify resistance-related mutations. Additionally, we analysed genomic differences among the three host strains, screening for anti-phage systems. Sequence analyses, PCR, and nuclease treatments of resistant host genomes and plasmids were used to assess the possibility of lysogeny and superinfection immunity. No single mutation or anti-phage system consistently explained FLiP resistance, as these varied across hosts. Resistant B114 contained FLiP genome in circular extrachromosomal dsDNA form, suggesting possible lysogeny. Surprisingly, we found that low quantities of FLiP sequences exist in bacterial populations not exposed to FLiP in the laboratory. This suggests that residing as extrachromosomal dsDNA elements in a small percentage of host cells may be a natural strategy for FLiP type phages to endure unfavourable conditions.

Abstract Copy number variations (CNVs) are a form of genetic alteration strongly implicated in numerous neurological and psychiatric disorders, as well as brain cancer. Replication stress is a common cause of CNVs. Despite the prevailing model that CNVs arise from DNA double strand breaks (DSBs), there has been no assay that directly perturbs presumed DSB sources and measures CNV output. Here, we identified a subset of recurrent DNA break clusters (RDCs) as a causal factor for CNV formation. In murine neural progenitor cells under replication stress, mapping the formation of CNVs revealed their location in RDC regions that contain actively transcribed genes. CRISPR/Cas9-mediated transcriptional suppression abrogated both RDC and CNV formation, but does not alter their replication timing. We found that DNA polymerase theta (Pol θ), a protector against CNV formation, plays a critical but context dependent role upstream of RDC formation. Chemically inhibiting the activity of Pol θ reduced end filling and micro-homology-mediated end joining in XRCC4/P53-deficient cells. Conversely, Pol θ inhibition led to elevated DSB density detection at RDC-containing loci in wild-type neural stem and progenitor cells, suggesting its role in preventing transcription-replication conflicts. Our data identify RDCs as contributors to genomic heterogeneity with plausible downstream effects on brain disorders and malignancy.

Fruiting bodies of mushroom-forming fungi (Agaricomycetes) exhibit the highest degree of multicellular complexity in fungi, yet the molecular underpinnings of their developmental programs remain incompletely understood. Here, we characterize gcd1 , a gene encoding a transcription factor in the Con7 subfamily of C2H2-type zinc finger proteins. This subfamily has previously been implicated in pathogenic morphogenesis in Ascomycota, but their role in Agaricomycetes has not previously been addressed. In Coprinopsis cinerea , CRISPR/Cas9-mediated deletion of gcd1 resulted in strains with severely impaired fruiting body morphogenesis, with malformed cap, stipe, and gill tissues. Gcd1 deletion strains lacked universal veil, resembling species with open (gymnocarpous) development. We find that GCD1/Con7 homologs are widely distributed in most Dikarya species and are mostly encoded by a single gene in each species’ genome. Transcriptome analyses identified several misregulated genes in the Δ gcd1 mutant, which pinpoint potential mechanisms underlying its developmental defects as well as provided insights into the morphogenesis of mushroom fruiting bodies. These findings establish GCD1 as a key regulator of multicellular development in C. cinerea and broaden the known functions of Con7-like transcription factors to include fruiting body morphogenesis in Agaricomycetes. Overall, our results and the morphogenetic role of Con7-like transcription factors of Ascomycota suggest functional conservation over half a billion years of evolution.

Abstract ERF/AP2 family transcription factors play crucial roles in plant growth, development, and stress responses. However, the functions of most family members remain unclear. Here, the role of ERF3 in Arabidopsis thaliana ​​ was investigated through the analysis of ​​CRISPR/Cas9-generated erf3 mutants and ERF3 -overexpressing plants​​. We found that ​​the erf3 mutants exhibited enhanced resistance, whereas ERF3-overexpressing (ERF3-OE) plants showed slightly reduced resistance to the bacterial pathogen Pst DC3000 compared with wild-type plants​​. Transcriptomic sequencing identified 674 differentially expressed genes (DEGs) between ​​the erf3 mutants and wild-type plants​​, including 134 upregulated genes (​​erf3up DEGs​​) and 540 downregulated genes (erf3down DEGs​​). The ​​erf3up DEGs were significantly enriched in defense-related processes, including SA pathway marker genes (PR2 and PR5)​​, whereas ​​the erf3down DEGs were enriched in hormone-responsive pathways, such as responses to JA, ethylene, SA, auxin, GA, and ABA​​. Interestingly, ​​most of these hormone-responsive genes are not involved in disease resistance but play important roles in growth, development, and abiotic stress responses​​. ​​ERF3 is induced by Pst DC3000, SA, and JA, and ERF3 proteins are enriched on the promoters of target genes harboring cis-acting elements (GCC or DRE boxes), such as PR5, IAA29, RAV2, BG1, LECRK-1.1, and AZI1, as demonstrated by ChIP analysis​​. ​​Overall, ERF3 functions as a negative regulator of the SA pathway in disease resistance and plays a critical role in balancing disease resistance with hormone-mediated growth and abiotic stress responses. Our work provides novel insights into the potential of exploiting ERF3 function to enhance plant disease resistance​​.

Immune checkpoint blockade has transformed cancer therapy, yet many patients fail to respond, and few new targets have emerged beyond PD-1 and CTLA-4. Alternative splicing dramatically diversifies the T cell proteome, but the functional roles of most isoforms remain unknown. Here we constructed the first single-cell splicing atlas of human CD8⁺ T cells, capturing dynamic isoform programs across activation and subset differentiation. This revealed distinct splicing footprints that refine conventional transcriptomic states and highlight receptor families with isoform-level regulation. To functionally interrogate these candidates, we developed SpliceSeek, a CRISPR-based pooled screening platform that perturbs splice sites to redirect isoform usage. Using SpliceSeek, we uncovered isoform-specific immune checkpoints whose perturbation enhanced effector function and tumor control, including the LRRN3-203 isoform, which augmented cytokine secretion and antitumor immunity in mice models. Together, our results establish alternative splicing as a targetable layer of immune regulation and demonstrate the potential of isoform-focused screening to expand the landscape of cancer immunotherapy.

Histone proteins and their variants have been found to play crucial and specialized roles in chromatin organization and the regulation of downstream gene expression; however, the relationship between histone sequence and its effect on chromatin organization remains poorly understood, limiting our functional understanding of sequence variation between distinct subtypes and across evolution and frustrating efforts to rationally design synthetic histones that can be used to engineer specified cell states. Here, we make the first advance towards engineered histone-driven chromatin organization. By expressing libraries of sequence variants of core histones in human cells, we identify variants that dominantly modulate chromatin structure. We further interrogate variants using a combination of imaging, proteomics, and genomics to reveal both cis and trans- acting mechanisms of effect. Functional screening with transcription factor libraries identifies transcriptional programs that are facilitated by engineered histone expression. Double mutation screens combined with protein language models allow us to learn sequence-to-function patterns and design synthetic histone proteins optimized to drive specific chromatin states. This work establishes a foundation for the high-throughput evaluation and engineering of chromatin-associated proteins and positions histones as tunable nodes for understanding and modulating mesoscale chromatin organization.

Current gene transfer methods often lack the precision, versatility, or efficiency when integrating large transgenes, limiting the ability to engineer therapeutic T-cells with more complex payloads. Here, we report ‘one-pot’ PASTA (Programmable and Site-specific Transgene Addition), a non-viral genome engineering strategy for large gene insertion that combines CRISPR-Cas-mediated homology-directed repair (HDR) and site-specific recombination via serine integrases. Using ‘one-pot’ PASTA with the Bxb1 integrase, we demonstrate efficient integration of transgenes at multiple genomic loci relevant for T-cell engineering (e.g., TRAC, B2M, CD3E, CD3Z, GAPDH ). For constructs > 8 kb, ‘one-pot’ PASTA outperforms conventional HDR by 19-fold on average and prime-editing-assisted site-specific integrase gene editing (PASSIGE) by 5-fold. This enables the delivery of multi-cistronic cargo to generate dual-antigen targeting CAR T-cells with a safety-switch that overcome antigen escape in lymphoma models. Finally, ‘one-pot’ PASTA can be further optimized with improved integrase enzymes, such as engineered variants of Pa01 or Bxb1, and plasmids with minimized backbones. In summary, ‘one-pot’ PASTA represents a versatile and scalable platform for precise, non-viral gene insertion in T-cells.

Summary Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) is the most established cellular conversion by exogenous master transcription factors (TFs). A deeper understanding of this yet inefficient process is critical to extending our capability to control cellular identity for medical applications. Here we report 14 genes essential for efficient iPSC generation, but dispensable for self-renewal. Of those, overexpression of Hic2 , a transcriptional suppressor highly expressed in PSCs, enhances iPSC generation ∼10-fold. This is achieved through a more direct transition towards pluripotency, bypassing an intermediate state with KLF4-dependent transient epidermal gene expression during iPSC generation. Mechanistically, HIC2 co-occupies these KLF4 targets and directly inhibits their expression. Our work demonstrates that master TFs necessary for cellular conversions can also activate obstructive genes during cellular reprogramming. We propose that identifying transcriptional suppressors against such side effects, like Hic2 , can be a powerful strategy to achieve more efficient TF-mediated cell conversions.

The tRNA nuclease SLFN11 is epigenetically silenced in ∼50% of treatment-naive tumours and is the strongest predictor of chemoresistance but why it is frequently inactivated in cancer is unknown. To acquire immortality, cancer cells can activate alternative lengthening of telomeres (ALT), typically accompanied by ATRX loss. Here, we implicate SLFN11 in sensing telomere replication stress, triggering eradication of ATRX deficient cells prior to ALT establishment. Whereas progressive telomere shortening of cells lacking telomerase and ATRX leads to telomere crisis and cell death, SLFN11 loss confers tolerance to PML-BLM dependent ALT intermediates, permitting emergence of ALT survivors. We propose that during tumorigenesis SLFN11 inactivation is selected as means to tolerate endogenous replication stress following telomere crisis, leading to the development of therapy resistant tumours before treatment.

Transcriptional regulatory proteins are frequent drivers of oncogenesis and common targets for drug discovery. The transcriptional co-activator, ENL, which is localized to chromatin through its acetyllysine-binding YEATS domain, is preferentially required for the survival and pathogenesis of acute leukemia. Small molecules that inhibit the ENL YEATS domain show anti-leukemia effects in preclinical models, which is thought to be caused by the downregulation of pro-leukemic ENL target genes. However, the transcriptional effects of ENL YEATS domain inhibitors have not been studied in models of intrinsic or acquired resistance and, therefore, the connection between proximal transcriptional effects and downstream anti-proliferative response is poorly understood. To address this, we identified models of intrinsic and acquired resistance and used them to study the effects of ENL YEATS domain inhibitors. We first discovered that ENL YEATS domain inhibition produces similar transcriptional responses in naive models of sensitive and resistant leukemia. We then performed a CRISPR/Cas9-based genetic modifier screen and identified in-frame deletions of the essential transcriptional regulator, PAF1, that confer resistance to ENL YEATS domain inhibitors. Using these drug-resistance alleles of PAF1 to construct isogenic models, we again found that the downregulation of ENL target genes is shared in both sensitive and resistant leukemia. Altogether, these data support the conclusion that the suppression of ENL target genes is not sufficient to explain the anti-leukemia effects of ENL antagonists.

The mature B cell compartment consists of follicular (FO) and marginal zone (MZ) B cells, which develop from transitional type 2 (T2) B cells and mount T-dependent and T-independent antibody responses, respectively. TACI, a member of the TNF receptor superfamily, is expressed on all mature B cells, with highest levels on MZ B cells and plasma cells. Previous studies reported that TACI is a negative regulator of B cell survival. However, this conclusion is confounded by elevated levels of BAFF, a cytokine that supports B cell survival, in TACI- deficient mice. We now show that TACI does not directly regulate B cell survival but rather has a cell-intrinsic role in MZ B cell development. Loss of TACI leads to reduced MZ B cell numbers and impaired T-independent antibody responses. Mechanistically, we show that TACI is required for MZ B cell development from T2 B cell precursors via activation of the PI3K-AKT pathway and subsequent inhibition of the FOXO1 transcription factor.