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.

Microridges are laterally elongated membrane protrusions from the apical surface of epithelial cells. Microridges are arranged in striking maze-like patterns. They are found on various mucosal epithelia in many animals, including the skin of zebrafish, where they are required to maintain mucus on the skin surface. Recent studies have revealed molecular mechanisms of how microridiges formation involving actin and actin-regulatory proteins. However, the molecular mechanism that deforms epithelial membranes to create microridge protrusions remain unknown. We have found that one of the I-BAR domain proteins, baiap2l1a, which is known to regulate membrane curvature, is required for microridge morphogenesis. CRISPR/Cas9 knockdown showed that baiap2l1a mutant zebrafish had defects in microridge morphogenesis. Baiap2l1a mutant zebrafish had shorter and wider microridges than WT microridges. Baiap2l1a localized to microridges, and its localization proceeded microridge actin formation. Furthermore, the baiap2l1a I-BAR domain, which binds and curves membranes, was sufficient to localize to microridges in zebrafish skin cells. Structure/function experiments revealed that the I-BAR domain alone could partially rescued microridge length in baiap2l1a mutants. A 39 amino acid deletion in the I-BAR domain, which caused the loss of one α-helix according to AlphaFold2 simulations, is sufficient to impair microridge localization and failed to rescue microridge elongation in baiap2l1a mutants. These results suggest that the I-BAR domain is required for baiap2l1a microridge localization and function. Eps8like1a, a member of the Eps8 family proteins known as an actin capping and bundling protein genetically interacted with baiap2l1a in microridge elongation. Together, we found that the membrane curvature protein baiap2l1a plays an important role in generating microridges in zebrafish epithelia.

ABSTRACT Proteins operate through a few critical residues, yet most proteins remain uncharacterized at the deep molecular resolution, particularly within essential genes, where functional dissection is obstructed by lethality. Here, we establish a platform for mutational scanning of essential genes at their endogenous locus , combining a repressible complementation system with multiplexed CRISPR-based genome editing in budding yeast. Our approach provides a generalizable framework for dissecting essential protein function in vivo , expanding the capacity to map critical residues underlying essential cellular processes. We applied this strategy to NRD1 , encoding an essential RNA Polymerase II (RNAPII) termination factor and performed a systematic alanine scanning with near-saturation coverage. We discovered novel and unexpected lethal mutations in the CTD-interacting domain (CID), thus revealing an unanticipated importance for this domain. Overall, our results demonstrate the power of our mutation scanning platform to map critical residues underlying essential cellular processes.

While most conserved microRNA (miRNA) transcripts harbor a suite of features that mediate their efficient biogenesis into small RNAs, some loci bear suboptimal attributes that enable additional layers of processing regulation. A notable example is cluster assistance, whereby a miRNA hairpin with suboptimal nuclear biogenesis can be enhanced by an optimal neighbor. This process involves local transfer of the Microprocessor complex, composed of the RNase III enzyme Drosha and its partner DGCR8, in concert with cofactors such as ERH and SAFB1/2. However, the mechanism(s) that underlie miRNA cluster assistance remain largely unclear. Here, we gain insights into this process by integrating mutant cells of Microprocessor and its cofactors with analysis of miRNA structure-function variants, biochemical tests and genomewide profiling. We define features of suboptimal miRNAs that render them dependent on cluster assistance, and distinguish amongst a network of proposed interactions amongst Microprocessor and its cofactors, to reveal a subset that are critical for cluster assistance. Most importantly, we use epistatic tests to separate and order the functional requirements for ERH and SAFB1/2 into a pathway. Our data indicate that ERH may engage in the process of Microprocessor transfer between hairpins, while SAFB factors (especially SAFB2) mediate recognition and stable binding of a suboptimal miRNA hairpin after Microprocessor transfer. Finally, we show how cluster assistance integrates into a feedback regulatory loop on Microprocessor, via Drosha-mediated cleavage of a suboptimal miRNA hairpin in the DGCR8 transcript. Altogether, our findings reveal complex regulatory transactions during biogenesis of clustered miRNAs.

SUMMARY DNA double-strand breaks and unresolved DNA replication intermediates are particularly dangerous during mitosis. Paradoxically, cells inactivate canonical DNA repair mechanisms during chromosome segregation in favor of alternative pathways that depend on TOPBP1 and CIP2A, but how they function is still poorly defined. Here, we describe the identification of DDIAS as a mitosis-specific DNA damage response protein. We establish DDIAS as a phosphorylation-dependent component and effector of the TOPBP1-CIP2A complex with single-stranded DNA (ssDNA)-binding activity, and thereby delineate a ssDNA protection mechanism that safeguards chromosome integrity during mitosis, particularly in BRCA-deficient cells. We also identify biallelic inactivating mutations in DDIAS and CIP2A in patients with severe neurodevelopmental phenotypes. These findings highlight the physiological importance of the DNA damage response in mitosis, and may open new avenues for synthetic-lethal therapy in BRCA-deficient cancers.

Leukocyte trafficking is a critical step in development of chronic intestinal diseases such as Crohn's disease. While strategies that block gut-homing have yielded partial success, this disease remains uncurable, leaving an unmet clinical need. This is the first paper to describe a role for cannabinoid receptor two (CB2R) signalling in promoting retinoic acid-mediated induction of the gut-homing associated integrin heterodimer α4β7. Using in vitro and in vivo models, we characterised the effects of pharmacological CB2R agonists and inverse agonists on T cell homing receptor expression and transmigration across gut-associated endothelial barriers. This ERK-dependent process coincides with increased T cell adherence in response to CB2R agonism with JWH133. These effects were reversed with an inverse agonist GP-1a in a CB2R-dependent manner. Selective deletion of CB2R using CRISPR in vitro or CD4Cre/+ floxed mice in vivo resulted in impaired endothelial cell adherence and decreased diapedesis into the ileal lamina propria. T cell-specific deletion of cnr2, the gene encoding CB2R, attenuated chronic murine ileitis characterised by decreased naïve T cell infiltration and loss of tissue architecture in 20wk TNFδARE/+ mice. This study supports further therapeutic development of CB2R-blocking drugs for the treatment of inflammatory bowel disease.

Micronutrients, particularly boron (B), iron (Fe), manganese (Mn), and zinc (Zn), are pivotal for cotton (Gossypium spp.) growth, reproductive success, and fiber quality, yet their roles are often overshadowed by macronutrient-focused fertility programs. This review synthesizes recent advancements in understanding the physiological, molecular, and agronomic roles of B, Fe, Mn, and Zn in cotton production, with a focus on their signaling integration and impact on nutrient use efficiency (NUE). Drawing from peer-reviewed liter-ature, experimental data, and regional surveys, we highlight how these micronutrients regulate critical processes such as photosynthesis, cell wall integrity, hormone signaling, and stress responses, directly in-fluencing root development, boll retention, and fiber quality. Deficiencies, exacerbated by soil pH, redox conditions, and nutrient interactions, contribute to significant yield gaps, even when macronutrients like nitrogen (N), phosphorus (P), potassium (K), and sulfur (S) are adequately supplied. Key genes, including BOR1, IRT1, NRAMP1, and GhZIP3, mediate micronutrient uptake and homeostasis, offering targets for breeding high-yield, nutrient-efficient cotton varieties. Advanced phenotyping using unmanned aerial ve-hicles (UAVs) and single-cell RNA sequencing (scRNA-seq) provide novel avenues for identifying nutri-ent-efficient genotypes and regulatory networks. The review also explores synergistic interactions between micronutrients and macronutrients to influence growth and yield of cotton. Future research directions in-clude leveraging microRNAs, CRISPR-based gene editing, and precision nutrient management to enhance B, Fe, Mn, and Zn use efficiency, addressing environmental challenges while closing persistent yield gaps in sustainable cotton production systems.

ABSTRACT CRISPR/Cas9 has transformed gene editing, enabling precise genetic modifications across species. However, existing sgRNA design prediction models based on in vitro data are difficult to generalize to in vivo contexts. In particular, approaches based on single-sgRNA design require additional filtering of in-frame mutations, which is inefficient in terms of both time and cost. In this study, we developed the first mammalian in vivo-trained prediction model to evaluate the efficiency of a dual-sgRNA-based exon deletion strategy. Using 230 editing outcomes of postnatal viable individuals, eight prediction models were constructed and evaluated based on generalized linear models and random forests. The final selected model, a Combined GLM, integrated the DeepSpCas9 score with k-mer sequence features, achieving an AUC of 0.759 (95% Confidence Interval: 0.697–0.821). Motif analysis revealed that CC sequences were associated with high efficiency and TT sequences were associated with low editing efficiency. This study demonstrates that integrating sequence-based features with existing design scores can improve sgRNA efficiency prediction in vivo. The proposed framework can be applied to the development of next-generation sgRNA design tools, with direct implications for gene therapy, effective animal model generation, and precision genome engineering. GRAPHICAL ABSTRACT

Background: Genetic mosaicism is a consequence of CRISPR-Cas9 genome editing that is difficult to study, especially when it involves structural variants occurring at low frequency. A comprehensive analysis of mosaicism requires deep and unbiased sequencing of the target loci, with accurate single molecule reads. Here we performed amplification-free PureTarget PacBio sequencing to investigate CRISPR-Cas9 outcomes at on-target and off-target sites in germline edited zebrafish and their offspring. Results: Thirty samples from pooled larvae and individual zebrafish were successfully sequenced, resulting in >1100x average target coverage. The PacBio reads reached an exceptional accuracy (QV39) over the target regions, with every read originating from a unique DNA molecule. The two haplotypes of the target loci displayed a balanced depth of coverage, while long-range PCR of the same samples resulted in skewed data. Further analysis of the PureTarget data revealed widespread genetic mosaicism in individual founder (F0) fish, with up to 18 distinct on-target events and 11 off-target events present in a single adult founder. Several CRISPR-Cas9 editing outcomes, including large structural variants and off-target mutations, were inherited to the F1 generation. Notably, as many as seven unique editing events were found among F1 juvenile offspring from a single founder pair, thereby confirming the presence of genetic mosaicism in germ cells of founder zebrafish. This implies that some consequences of CRISPR-Cas9 editing may emerge only in the second generation. We also analyzed DNA methylation but did not observe altered 5mC CpG signals in genome edited samples. Conclusions: PureTarget enables efficient, accurate and unbiased analysis of the full spectrum of genetic mosaicism in a sample. The potential risks of introducing germ cell mosaicism in founder individuals should be carefully evaluated when designing germline genome editing experiments.