Human homolog of Ariadne (HHARI) is a RING-between-RING ubiquitin E3 ligase which interacts with cullin-RING E3 ligase (CRL) complexes. HHARI has been implicated in the type-I interferon anti-viral response. However, how HHARI drives interferon signalling is not fully understood and the function of the unique, highly conserved acidic N-terminal domain of the protein is unknown. Here, we show that HHARI stimulates interferon-β secretion and autocrine type-I interferon signalling by directly targeting the viral RNA sensor RIG-I (Retinoic Acid-Inducible Gene I) in a neddylation-dependent manner. This suggests that neddylation inhibition could be used to treat interferonopathies and related diseases. Truncated HHARI containing only the N-terminal acidic/UBA-like domains retained the ability to induce interferon signalling in a neddylation-dependent mechanism. HHARI-mediated interferon-β secretion was enhanced by overexpression of cullins 1-5. The N-terminal acidic/UBA-like domain of HHARI is critical for RIG-I activation and interferon signalling, as removal of these domains inactivated the pro-interferon phenotype. We propose a mechanism by which the N-terminus of HHARI interacts with all neddylated cullins leading to endogenous HHARI activation. This suggests a model in which the N-terminus of HHARI ‘unlocks’ and activates neddylated cullins, which in turn are required for activation of HHARI itself. As cullins typically form modular cullin-RING ligase super-assemblies our findings imply that the HHARI N-terminus domain is a critical regulator of the versatile CRL system, which, through widespread protein ubiquitylation, controls many eukaryotic cell functions.

High-throughput phenotypic screening has historically relied on manually selected features, limiting our ability to capture complex cellular processes, particularly neuronal activity dynamics. While recent advances in self-supervised learning have revolutionized the ability to study cellular morphology and transcriptomics, dynamic cellular processes have remained challenging to phenotypically profile. To address this limitation, we developed Plexus, a novel self-supervised model specifically designed to capture and quantify network-level neuronal activity dynamics. Unlike existing phenotyping tools that focus on static readouts, Plexus leverages a novel network-level cell encoding method, which enables it to efficiently encode dynamic neuronal activity data into rich representational embeddings. In turn, Plexus achieves state of the art performance in detecting phenotypic changes in neuronal activity. We validated Plexus using a comprehensive GCaMP6m simulation framework and demonstrated its enhanced ability to classify distinct neuronal activity phenotypes compared to traditional signal-processing approaches. To enable practical application, we integrated Plexus with a scalable experimental system utilizing human iPSC-derived neurons equipped with the GCaMP6m calcium indicator and CRISPR interference machinery. This integrated platform successfully identified nearly twice as many distinct phenotypic changes in response to genetic perturbations compared to conventional methods, as demonstrated in a 52-gene CRISPRi screen across multiple iPSC lines. Using this framework, we identified potential genetic modifiers of aberrant neuronal activity in frontotemporal dementia, illustrating its utility for understanding complex neurological disorders.

ABSTRACT Zebrafish serve as a valuable model organism for studying human genetic diseases. While generating knockout lines is relatively straightforward, introducing precise disease-specific genetic variants by knock-in (KI) remains challenging. KI lines, however, enable more accurate studies of molecular and physiological consequences of genetic diseases. Their generation is often hampered by low editing efficiencies (EE) and potential off-target effects. In this study, we optimized conventional CRISPR/Cas9-mediated homology-directed repair (HDR) strategies for precise KI of genetic variants in zebrafish and compared their efficacy with prime editing (PE), a recently developed technique that is not yet commonly used. Using next-generation sequencing (NGS), we determined KI EE by HDR for six unique base-pair substitutions in three different zebrafish genes. We assessed the effect of 1) varying Cas9 amounts, 2) HDR templates with chemical modifications to improve integration efficiency, 3) different micro-injection procedures, and 4) synonymous guide-blocking variants in the protospacer sequence. Increasing Cas9 amounts augmented KI EE, with optimal injected amounts of Cas9 between 200 and 800 pg. The use of Alt-R™ HDR templates (IDT) further increased KI EE, while guide-blocking modifications did not. Injecting components directly into the cell was not superior to injections into the yolk. PE, however, increased EE up to fourfold and expanded the F0 founder pool for four targets when compared to conventional HDR editing, with fewer off-target effects. Therefore, PE is a very promising methodology for improving the creation of precise genomic edits in zebrafish, facilitating the modeling of human diseases.

Advances in single-cell sequencing and CRISPR technologies have enabled detailed case-control comparisons and experimental perturbations at single-cell resolution. However, uncovering causal relationships in observational genomic data remains challenging due to selection bias and inadequate adjustment for unmeasured confounders, particularly in heterogeneous datasets. To address these challenges, we introduce causarray, a doubly robust causal inference framework for analyzing array-based genomic data at both bulk-cell and single-cell levels. causarray integrates a generalized confounder adjustment method to account for unmeasured confounders and employs semiparametric inference with flexible machine learning techniques to ensure robust statistical estimation of treatment effects. Benchmarking results show that causarray robustly separates treatment effects from confounders while preserving biological signals across diverse settings. We also apply causarray to two single-cell genomic studies: (1) an in vivo Perturb-seq study of autism risk genes in developing mouse brains and (2) a casecontrol study of Alzheimer’s disease using three human brain transcriptomic datasets. In these applications, causarray identifies clustered causal effects of multiple autism risk genes and consistent causally affected genes across Alzheimer’s disease datasets, uncovering biologically relevant pathways directly linked to neuronal development and synaptic functions that are critical for understanding disease pathology.

The detection of cytosolic dsDNA is tightly regulated to avoid pathological inflammatory responses. A major pathway involved in their detection relies on the cyclic GMP-AMP synthase (cGAS) that triggers activation of the Stimulator of interferon genes (STING) which subsequently drives the expression of inflammatory genes and type I Interferons (IFNs). Here, we show that the methyl-CpG-binding protein 2 (MECP2), a major transcriptional regulator, controls dsDNA-associated inflammatory responses. We show that the presence of cytosolic dsDNA promotes MECP2 export from the nucleus to the cytosol where it interacts with dsDNA, dampening cGAS activation. Our data also indicate that MECP2 export from the nucleus partially phenocopies MECP2 deficiency, leading to the expression of inflammatory and interferon stimulated genes, enforcing an antiviral state. Finally, we also show that MECP2 displacement from the nucleus following dsDNA stimulation is sufficient to disrupt its canonical function, leading to the reactivation of otherwise repressed genes, such endogenous retroelements of the Long interspersed nuclear element-1 (LINE-1) family. Re-expression of the latter led to the accumulation of DNA species feeding cGAS-dependent signaling and can be dampened by reverse transcriptase inhibitors. We thus establish a previously unforeseen direct role of MECP2 in the regulation of the breadth and nature of dsDNA-associated inflammatory responses. Furthermore, our results suggest that targeting dsDNA-associated pathways or pharmacological inhibition of LINE-1 may bear therapeutic hopes for Rett syndrome (RTT) patients that present with MECP2 deficiency.

Summary Base editing stands at the forefront of genetic engineering, heralding precise genetic modifications with broad implications. While CRISPR-based DNA and RNA base editing systems capitalize on sgRNA-guided specificity and diverse deaminase functionalities, the pursuit of efficient C-to-U RNA editing has been hampered by the inherent constraints of cytidine deaminases. Here, we report an RNA base editing platform by refining cytidine deaminases, termed professional APOBECs (ProAPOBECs), through systematic enhancements and AI-driven protein engineering. ProAPOBECs demonstrate unprecedented catalytic versatility, particularly fused with RNA-recognizing Pumilio and FBF (PUF) proteins. We present the first effective use of RNA base editing in the brain with ProAPOBECs in Mef2c mutant mice, a model for autism. The AAV-mediated RNA base editing via ProAPOBEC not only corrects genetic mutation in mRNAs but significantly alleviates autistic-like behaviors in the mice. This work introduces a pioneering collection of RNA base editing instruments, emphasizing their therapeutic potential in combatting genetic disorders.

Recent advances in functional genomics tools have ushered in a new era of genetic editing to identify molecular pathways relevant to developmental and disease biology. However, limited model systems are available that adequately mimic cell states and phenotypes associated with human disease pathways. Here, we quantitatively analyzed the founder population bottleneck effect and demonstrated how the population changes from induced pluripotent stem cells (iPSCs) to hematopoietic stem cells and to the final induced macrophage population. We then engineered SAMHD1 knockout (KO) iPSC and characterized the iPSC line with RNA Seq, and induced macrophages from two distinct protocols with functional analysis. We then generated SAMHD1 KO CRISPR-dCAS9 KRAB iPSC through lenti-viral transduction aiming to increase the efficiency of lentiviral mediated gene transfer. We demonstrated increased lenti-viral transduction efficiency in induced macrophage, as well as microglia induced with two distinct protocols. This model allows for efficient gene knock down, as well as large-scale functional genomics screens in mature iPSC-derived macrophages or microglia with applications in innate immunity and chronic inflammatory disease biology. These experiments highlight the broad applicability of this platform for disease-relevant target identification and may improve our ability to run large-scale screens in iPSC-derived myeloid model systems.

Centromeres are chromosomal loci that ensure proper chromosome segregation by providing a platform for kinetochore assembly and spindle force transduction during cell division. Human centromeres are defined primarily by a unique chromatin domain featuring the histone H3 variant, Centromere Protein A (CENP-A), that marks a single active centromere locus per chromosome. CENP-A chromatin typically occupies a small subregion of low DNA methylation within multi-megabase arrays of hypermethylated alpha-satellite repeats and constitutive pericentric heterochromatin. However, the mechanisms defining and maintaining precise centromere position and domain size, and the role of the underlying alpha satellite DNA sequence, are poorly characterised. Using an experimentally-induced neocentromere in RPE1 cells, we discovered that the SUV39H1 and H2 methyltransferases tri-methylate H3K9 at neocentromere boundaries to maintain CENP-A domain size independent of DNA methylation or satellite sequences. Furthermore, we found that the CENP-A domain at canonical alpha-satellite-based centromeres is characterized by local depletion of H3K9me3-mediated heterochromatin, coinciding with the DNA methylation dip region. We identified the SETDB1 methyltransferase as key to maintaining H3K9me3 within flanking active higher order alpha satellite arrays while SUV39s and SUZ12 contribute to globally heterochromatinize both alpha satellites and neighbouring repeats. Loss of this heterochromatin boundary results in the progressive expansion of the primary CENP-A domain, erosion of DNA methylation, and the nucleation of new centromeres across alpha satellite sequences. Our study identifies the functional specialization of different H3K9 methyltransferases across centromeric and pericentric domains, crucial for maintaining centromere domain size and number.

Drug discovery requires a deep understanding of disease mechanisms, making the integration and analysis of diverse, multi-modal data types essential. These data types, spanning omics types, disease- associated, and biological pathway information, often originate from disparate sources and must be combined to uncover critical insights. We have developed iPANDDA ( in-silico Pipeline for Agnostic Network-based Drug Discovery Analysis ), a computational pipeline that integrates multi-modal data through a network-based approach to predict candidate drug target proteins for specific diseases. We applied iPANDDA to lung squamous cell carcinoma (LUSC), a subtype of non-small cell lung cancer (NSCLC) that accounts for approximately 25% of all lung cancer cases globally. Despite advances in cancer therapeutics, targeted therapies specifically approved for LUSC remain lacking, exacerbated by the shortage of robust models for studying LUSC carcinogenesis and therapeutic responses. The SOX2 gene, amplified in about 50% of LUSC patients, plays a critical role in driving and maintaining the cancer phenotype. Using iPANDDA, we identified relevant therapeutic targets for SOX2-dependent LUSC. Selected candidate drug targets were validated in vitro using cell-based models. We conducted target inhibition studies in both SOX2-dependent and non-SOX2-dependent cell lines, evaluating the effects of inhibition and knockout through cell viability assays. Our findings confirmed key monotherapy and combination therapy targets for SOX2-focused models. Specifically, we validated the AKT and mTOR complexes as promising therapeutic targets for LUSC. Additionally, we identified potential pathways for developing novel combination therapies targeting SOX2-dependent LUSC. iPANDDA offers a robust approach to refining and focusing therapeutic strategies for diseases with unmet clinical needs.

Transfer RNA molecules have been recently recognized as widespread targets of bacterial immune systems. Translation inhibition through tRNA cleavage or modification inhibits phage propagation, thereby protecting the bacterial population. To counteract this, some viruses encode their own tRNA molecules, allowing infection to take place. The AriB effector of the PARIS defence system is a Toprim nuclease previously shown to target the E. coli tRNA Lys(UUU) , but not a tRNA Lys(UUU) variant encoded by bacteriophage T5. We demonstrate here that the T5-tRNA Lys(UUU) is required but not sufficient to bypass PARIS immunity. Combining tRNA-sequencing, genetics, phage infection and in vitro biochemical data, we reveal that the E. coli tRNA Thr(UGU) , is another prime target of AriB and tRNA Asn(GUU) represents a secondary, yet biologically relevant, target of the PARIS effector. Activated AriB protein cleaves these targets in vitro , and the cleavage reaction is not dependent on the presence of specific tRNA modifications. We show that the overexpression of phage T5 tRNA Lys(UUU) , tRNA Thr(UGU) and tRNA Asn(GUU) variants is sufficient to inhibit PARIS anti-viral defence. Finally, we propose a model for tRNA recognition by the AriB dimer and provide molecular details of its nuclease activity and specificity.

Commander is an endosome associated sixteen protein assembly that associates with the sorting nexin 17 (SNX17) cargo adaptor to regulate cell surface recycling of internalised integral membrane proteins including integrins and lipoprotein receptors. Mutations in Commander are causative for Ritscher-Schinzel syndrome (RSS), a multiorgan developmental disease associated with a core triad of cerebellar-cardiac-craniofacial malformation. Here, using unbiased proteomics and computational modelling, we identify leucine rich melanocyte differentiation associated (LRMDA) as a novel Commander binding protein. Using recombinant protein reconstitution, we show that LRMDA simultaneously associates with Commander and active RAB32, and, by revealing that LRMDA and SNX17 share a common mechanism of Commander association, establish the mutually exclusive nature of RAB32-LRMDA-Commander and SNX17-Commander assemblies. From functional analysis in human melanocytes, we establish an essential role for RAB32-LRMDA-Commander in melanosome biogenesis and pigmentation and reveal a distinct functional role for SNX17-Commander in this organelle biogenesis pathway. We reveal how LRMDA mutations, causative for oculocutaneous albinism type 7 (OCA7), a hypopigmentation disorder accompanied by poor visual acuity, uncouple RAB32 and Commander binding thereby establishing the mechanistic basis of this disease. Our discovery and characterisation of this alternative Commander assembly establishes an unrecognised plasticity of Commander function within a highly complex organelle biogenesis pathway. This extends Commander function beyond the confines of SNX17-mediated cell surface recycling into RAB32-family mediated biogenesis of lysosome-related organelles and, potentially, other RAB32 regulated pathways including host-pathogen defence mechanisms. Our work also extends the breath of Commander pathway dysfunction for human disease.

ABSTRACT The endosomal-lysosomal network is a hub of organelles that orchestrate the dynamic sorting of hundreds of integral membrane proteins to maintain cellular homeostasis. VPS29 is a central conductor of this network through its assembly into Retromer, Retriever and Commander endosomal sorting complexes, and its role in regulating RAB GTPase activity. Two VPS29 isoforms have been described, VPS29A and VPS29B, that differ solely in their amino-terminal sequences. Here we identify a third VPS29 isoform, which we term VPS29C, that harbours an extended amino-terminal sequence compared to VPS29A and VPS29B. Through a combination of AlphaFold predictive modelling, in vitro complex reconstitution, mass spectrometry and molecular cell biology, we find that the amino-terminal VPS29C extension constitutes an autoinhibitory sequence that limits access to a hydrophobic groove necessary for effector protein recruitment to Retromer, and association with Retriever and Commander. VPS29C is therefore unique in its ability to uncouple Retromer-dependent cargo sorting from the broader roles of VPS29A and VPS29B in regulating the endosomal-lysosomal network through accessory protein recruitment. Our identification and characterisation of VPS29C points to additional complexity in the differential subunit assembly of Retromer, an important consideration given the increasing interest in Retromer as a potential therapeutic target in neurodegenerative diseases. SIGNIFICANCE STATEMENT The endosomal-lysosomal network is essential for normal cellular function with network defects being associated with numerous neurodegenerative diseases. Two heterotrimeric complexes, Retromer and Retriever, control transmembrane protein recycling through the network. Of these, reduced Retromer expression is observed in Alzheimer’s disease and Retromer mutations lead to familial Parkinson’s disease. Here, we identify and characterise a new isoform of VPS29, a subunit shared between Retromer and Retriever. We reveal how this isoform, VPS29C, adopts an auto-inhibitory conformation to limit its association into Retriever and restrict the binding of VPS29C-containing Retromer to accessory proteins vital for regulating network function. By revealing added complexity in Retromer assembly and function, we provide new insight into Retromer’s potential as a therapeutic target in neurodegenerative diseases.

ABSTRACT Tomato ( Solanum lycopersicum ) is a climacteric fruit displaying a peak of respiration at the onset of ripening accompanied by increased synthesis of ethylene and carotenoid pigments. Chromoplast and mitochondrial respiration participate at different stages of fruit ripening, but their in vivo regulation and function remains unclear. We determined the in vivo activities of the mitochondrial alternative oxidase (AOX) and cytochrome oxidase pathways and quantified the levels of respiratory- and ripening-related gene transcripts, primary metabolites and carotenoids in ripening tomato fruits with or without a functional chromorespiration. Furthermore, we carried out physiological, molecular and metabolic analyses of CRISPR-Cas9 mutants defective in AOX1a, the main AOX isoform up-regulated during tomato fruit ripening. We confirmed that PTOX-dependent chromorespiration is only relevant at late stages of ripening and found that in vivo AOX activity significantly increased at the breaker stage, becoming the main contributor to climacteric respiration when ripening is unleashed. This activation did not correlate with gene expression but was likely due to increased levels of AOX activators such as pyruvate (a metabolic precursor of carotenoids), 2-oxoglutarate and succinate. A strong alteration of ripening-related metabolites was observed in aox1a mutant fruits, highlighting a key role of the AOX pathway at the onset of ripening. Our data suggest that increased supply of TCA cycle intermediates at climacteric stage allosterically enhance AOX activity, thus allowing the reoxidation of NAD(P)H to ensure carbon supply for triggering ethylene and carotenoid biosynthesis.

In this study, we examined the role of AKAP12, in endothelial cell motility, with a specific focus on AKAP12 variants AKAP12v1 and AKAP12v2. Previous work has shown that AKAP12, a multivalent A-kinase anchoring protein that binds to PKA and several other proteins regulating protein phosphorylation, is expressed at low levels in most endothelia in vivo but at higher levels in cells in vitro . Here, we found that AKAP12 expression in endothelial cell (HUVEC) cultures was cell density-dependent, with the expression being highest in subconfluent cultures and lowest in confluent cultures. AKAP12 expression was also elevated in cells at the wound edge of wounded endothelial cell monolayers. Knockdown of variants 1 and 2 inhibited cell migration, whereas CRISPR/Cas9 knockout of AKAP12v1 enhanced migration, indicating that the absence of this variant and the presence of AKAP12v2 may shift the signaling pathways. Further analysis using bulk RNA sequencing revealed that the loss of AKAP12v1 affects genes associated with cell migration and intercellular junctions. We propose that AKAP12v1 and AKAP12v2 work together to modulate endothelial cell migration, providing insights into their distinct yet complementary roles in endothelial function and potential implications for cardiovascular health.

ABSTRACT The use of genetically modified non-conventional yeast provides significant potential for the bioeconomy by diversifying the tools available for the development of sustainable and novel products. In this study, we sequenced and annotated the genome of Kluyveromyces marxianus Y-1190 to establish it as a platform for lactose valorization. The strain was chosen for rapid growth on lactose-rich dairy permeate, high transformation efficiency, and ease of culturing in bioreactors. Genomic sequencing revealed that K. marxianus Y-1190 possesses single nucleotide polymorphisms associated with efficient lactose metabolism. The strain is diploid with notable genomic heterogeneity, which appears to be critical for its robust growth and acid tolerance. To further exploit this platform strain, we developed protocols for gene and chromosome manipulation using CRISPR editing, constructed and validated a series of promoters compatible with MoClo vectors, and designed synthetically inducible promoters for K. marxianus . These tools enable precise control over gene expression, allowing for the tailored optimization of metabolic pathways and production processes. The synthetic promoters provide flexibility for dynamic expression tuning, while the CRISPR-based editing protocols facilitate targeted genetic modifications with high efficiency. Together, these advancements significantly enhance the genetic toolbox for K. marxianus , positioning it as a versatile platform for industrial biotechnology. These tools open new opportunities for the sustainable production of bio-based chemicals, fuels, and high-value products, leveraging lactose-rich feedstocks to contribute to a circular economy.

The application of CRISPR-based genome editing tools in plants is often challenged by low editing efficiencies, requiring most plant editing workflows to proceed through the delivery of pre-formed ribonucleoprotein (RNP) complexes to protoplasts. Here, we report increases in protoplast-based RNP delivery and genome editing efficiencies through the addition of anionic polymers to standard protoplast transfection protocols. We test addition of various polymers and peptides for their ability to increase genome editing efficiencies in both Nicotiana benthamiana and Arabidopsis thaliana protoplasts, by adding these components to standard PEG-based protoplast transfection protocols: i) non-covalent addition of charged polymers, ii) non-covalent addition of amphiphilic peptide A5K, and iii) tyrosinase-mediated covalent conjugation of various relevant peptide motifs directly to the RNP. Incorporation of the amphiphilic peptide A5K or covalent attachment of peptides to the RNP had no positive effect on editing efficiencies. However, we found that addition of anionic polymer polyglutamic acid to standard PEG transfection protocols significantly improved editing efficiencies in both Nicotiana benthamiana and Arabidopsis thaliana protoplasts relative to RNPs alone without negatively impacting protoplast viability. Our results suggest anionic polymers stabilize the RNP and increase the colloidal stability of the protoplast transfection workflow. This simple and straightforward method of stabilizing Cas9 RNPs can be easily adopted by others working on direct protein delivery to plant protoplasts to increase genome editing efficiencies. Key message The addition of anionic polymer polyglutamic acid to standard protoplast PEG transfection workflows enhance CRISPR-Cas9-mediated gene editing in plant protoplasts. Our results suggest the mechanism of increased transfection efficiency is due to colloidal stabilization of RNPs. Graphical Abstract

Mutations in the FUS gene cause aggressive and often juvenile forms of amyotrophic lateral sclerosis (ALS-FUS). In addition to mRNA, the FUS gene gives rise to a partially processed RNA with retained introns 6 and 7. We demonstrate that these FUSint6&7-RNAs form nuclear condensates scaffolded by the highly structured intron 7 and associated with nuclear speckles. Using hybridization-proximity labelling proteomics, we show that the FUSint6&7-RNA condensates are enriched in splicing factors and the m6A reader YTHDC1. These ribonucleoprotein structures facilitate post-transcriptional FUS splicing and depend on m6A/YTHDC1 for their maintenance. FUSint6&7-RNAs become hypermethylated in cells expressing mutant FUS, leading to their enhanced condensation and consequently, splicing. We further demonstrate that FUS protein is repelled by m6A. Thus, ALS-FUS mutations may cause an abnormal activation of FUS post-transcriptional splicing via altered RNA methylation. Strikingly, ectopic expression of FUS intron 6&7 sequences dissolves the endogenous FUSint6&7-RNA condensates, downregulating FUS mRNA and protein. Overall, we describe an RNA condensation-dependent mechanism regulating FUS splicing that can be harnessed for developing new therapies.

FISHNET uses prior biological knowledge, represented as gene interaction networks and gene function annotations, to identify genes that do not meet the genome-wide significance threshold but replicate nonetheless. Its input is gene-level P-values from any source, including omicsWAS, aggregation of GWAS P-values, CRISPR screens, or differential expression analysis. It is based on the idea that genes whose P-values are low due to sampling error are distributed randomly across networks and functions, so genes with suggestive P-values that cluster in densely connected subnetworks and share common functions are less likely to reflect sampling error and more likely to replicate. FISHNET combines network and function analysis with permutation-based P-value thresholds to identify a small set of exceptional genes that we call FISHNET genes. Applied to 11 cardiovascular risk traits, FISHNET identified 19 gene-trait relationships that missed genome-wide significance thresholds but, nonetheless, replicated in an independent cohort. The replication rate of FISHNET genes matched or exceeded that of other genes with similar P-values. FISHNET identified a novel association between RUNX1 expression and HDL that is supported by experimental evidence that RUNX1 promotes white fat browning, which increases HDL cholesterol levels. FISHNET also identified an association between LTB expression and BMI that is supported by experimental evidence that higher LTB expression increases BMI via activation of the LTβR pathway. Both associations failed genome-wide significance thresholds, highlighting FISHNET’s ability to uncover meaningful relationships missed by traditional methods. FISHNET software is freely available at https://doi.org/10.5281/zenodo.14765850 .

Immunotherapy is now standard of care for multiple myeloma, where the most common targets are B cell maturation antigen, CD38, and G protein-coupled receptor class C group 5 member D but strategies to identify additional targets are needed. We have utilized two large datasets of genomic data and integrated them with existing databases to identify expressed cell surface targets in myeloma patients. Importantly, we also identify targets specific to genomic defined subgroups of patients including primary translocations and high-risk subgroups. Examples of subgroup targets include ROBO3 in t(4;14), CD109 in t(14;16), CD20 in t(11;14), GPRC5D in 1q+, and ADAM28 in biallelic TP53 samples. Expression was validated by flow cytometry and CRISPR-Cas9 knock out models. Sub-clonal differences in expression were noted, as was alternative splicing of existing immunotherapy targets such as FCRL5 . These results highlight the use of genomic stratification to identify novel therapeutic targets. Graphical Abstract

Multiple myeloma (MM) is a neoplasm of antibody-producing plasma cells and is the second most prevalent hematological malignancy worldwide. Development of drug resistance and disease relapse significantly impede the success of MM treatment, highlighting the critical need to discover novel therapeutic targets. In a custom CRISPR/Cas9 screen targeting 197 DNA damage response-related genes, Protein Arginine N-Methyltransferase 1 (PRMT1) emerged as a top hit, revealing it as a potential therapeutic vulnerability and survival dependency in MM cells. PRMT1, a major Type I PRMT enzyme, catalyzes the asymmetric transfer of methyl groups to arginine residues, influencing gene transcription and protein function through post-translational modification. Dysregulation or overexpression of PRMT1 has been observed in various malignancies including MM and is linked to chemoresistance. Treatment with the Type I PRMT inhibitor GSK3368715 resulted in a dose-dependent reduction in cell survival across a panel of MM cell lines. This was accompanied by reduced levels of asymmetric dimethylation of arginine (ADMA) and increased arginine monomethylation (MMA) in MM cells. Cell cycle analysis revealed an accumulation of cells in the G0/G1 phase and a reduction in the S phase upon GSK3368715 treatment. Additionally, PRMT1 inhibition led to a significant downregulation of genes involved in cell proliferation, DNA replication, and DNA damage response (DDR), likely inducing genomic instability and impairing tumor growth. This was supported by Reverse Phase Protein Array (RPPA) analyses, which revealed a significant reduction in levels of proteins associated with cell cycle regulation and DDR pathways. Overall, our findings indicate that MM cells critically depend on PRMT1 for survival, highlighting the therapeutic potential of PRMT1 inhibition in treating MM.

Abstract Cas6 family ribonucleases can generate functional crRNAs in most type I and type III CRISPR-Cas systems. Most existing studies of Cas6 functions are mainly focused on nuclease activity in vitro and Cas6-processed product characterization in vivo. However, the biological functions of co-occurrence and cross-cleavage of various Cas6 proteins in a multi-CRISPR system host remain largely unexplored. In this study, we biochemically characterized the cross-cleavage of two Cas6 proteins in Thermus thermophilus HB27 and found for the first time that Cas6 could anchor the mature crRNA and interact with Cas5 subunit of type I-B system, revealing the functions of Cas6 to mediate the assembly of the type I Cascade complex. We further demonstrated that Cas6 of type III CRISPR-Cas system could be assembled into I-B Cascade complex, in turn significantly inhibiting the interference activity of type I-B system as an anti-CRISPR. Our findings provide an insight into the functional coupling and regulation mechanisms underlying multiple CRISPR-Cas systems, thereby offering valuable references for designing and controlling type I CRISPR-based gene editing tools precisely.

The plant cell wall not only serves as a physical barrier against pathogens but, when damaged, also functions as a source of cell wall-derived molecules that play crucial roles in plant immunity as damage-associated molecular patterns (DAMPs). While oligogalacturonides from homogalacturonan are well-studied DAMPs, the immune-signaling potential of other cell wall components remains largely unexplored. Conventional genetic and biochemical approaches aimed at identifying ligand-receptor pairs in plant immunity have been limited by the vast diversity of potential ligand molecules and functional redundancy of putative receptors. Here, we developed a high-throughput screening pipeline that simultaneously examines multiple interactions between plant cell wall-derived glycans and >350 extracellular domains (ECDs) of receptor kinases and receptor like proteins in Arabidopsis, resulting in the screening of >40,000 interactions. We discovered a group of leucine-rich repeat receptor kinases named ARMs (AWARENESS of RG-I MAINTENANCES) that interact with rhamnogalacturonan-I (RG-I), a major component of pectin. RG-I treatment induced pattern-triggered immunity responses, with distinct kinetics compared to oligogalacturonide responses. We identified RG-I oligosaccharide structures required for interaction with ARM receptors and immune activation, and found that ARM receptors are redundantly involved in plant immunity. Thus, our approach provides a powerful platform for discovering glycan-receptor pairs in plants.

ABSTRACT Primary cilia have been considered tumor-suppressing organelles in cholangiocarcinoma (CCA), though the mechanisms behind their protective role are not fully understood. This study investigates how the loss of primary cilia affects DNA damage response (DDR) and DNA repair processes in CCA. Human cholangiocyte cell lines were used to examine the colocalization of DNA repair proteins at the cilia and assess the impact of experimental deciliation on DNA repair pathways. Deciliation was induced using shRNA knockdown or CRISPR knockout of IFT20, IFT88, or KIF3A, followed by exposure to the genotoxic agents cisplatin, methyl methanesulfonate (MMS), or irradiation. Cell survival, cell cycle progression, and apoptosis rates were evaluated, and DNA damage was assessed using comet assays and γH2AX quantification. An in vivo liver-specific IFT88 knockout model was generated using Cre/Lox recombination. Results showed that RAD51 localized at the cilia base, while ATR, PARP1, CHK1 and CHK2 were found within the cilia. Deciliated cells displayed dysregulation in critical DNA repair. These cells also showed reduced survival and increased S-phase arrest after genotoxic challenges as compared to ciliated cells. Enhanced DNA damage was observed via increased γH2AX signals and comet assay results. An increase in γH2AX expression was also observed in our in vivo model, indicating elevated DNA damage. Additionally, key DDR proteins, such as ATM, p53, and p21, were downregulated in deciliated cells after irradiation. This study underscores the crucial role of primary cilia in regulating DNA repair and suggests that targeting cilia-related mechanisms could present a novel therapeutic approach for CCA. New and Noteworthy: Our findings reveal a novel connection between primary cilia and DNA repair in cholangiocytes. We showed that DDR and DNA repair proteins localize to cilia, and that deciliation leads to impaired cell survival and S-phase arrest under genotoxic stress. Deciliated cells exhibit heightened DNA damage, evidenced by increased γH2AX signals and comet assay results, a phenotype mirrored in in vivo IFT88 knockout mice. Furthermore, key DDR regulators, including ATM, p53, and p21, are downregulated in deciliated cells following irradiation, highlighting a crucial role for primary cilia in maintaining genome stability.

SUMMARY Prostate cancer (PCa) is a prevalent male cancer with high survival rates, except in advanced or metastatic stages, for which effective treatments are lacking. Metastatic PCa involves complex mechanisms including loss of tumor suppressor genes and DNA repair molecules, which impacts therapy responses. We have reanalyzed data from a CRISPR/Cas9 genome wide screening previously performed to identify essential regulators of invasive abilities of the metastatic cell line DU145 identifying SYCP3 as a regulator of metastatic invasion. Subsequent analyses of tumor samples demonstrated that SYCP3 expression is frequently upregulated in PCa tumors from patients in advanced stages. Furthermore, SYCP3 genetic depletion significantly reduced the invasive and migratory abilities of DU145 cells and increased their adhesion capacity. Additionally, and due to the implication of SYCP3 on DNA repair processes, we have analyzed the role of SYCP3 on the cellular response to radiotherapy (RT) and found that its depletion induced RT resistance, suggesting a role for SYCP3 in DNA damage response and genomic instability. All these data support a role for SYCP3 in PCa metastasis and provides opportunities for personalized medicine.

Genetically engineered T-cell therapies rely heavily on genome editing tools, such as the CRISPR/Cas9 system. However, unintended on-target chromosomal alterations, including large deletions and chromosome loss can occur and pose significant risks including tumorigenesis. Here we combined CasPlus and optimized guide RNAs to reduce these issues in CRISPR/Cas9 engineering human primary T cells. CasPlus, which integrates an engineered T4 DNA polymerase with Cas9 nuclease and guide RNA, promotes favorable small insertions (1-2 bp) while reducing large deletions and chromosome loss in T cells. Our optimized guide RNAs favoring small insertions reduced large deletions and chromosome loss by two- to five-fold versus those favoring small deletions. Moreover, combining optimized guide RNA with T4 DNA polymerase further synergistically reduced large deletions and chromosome loss by additional two-fold. Notably, replacing currently used guide RNA pairs in clinically applications with optimized pairs biased towards small insertions, along with CasPlus instead of Cas9, for editing greatly reduced large deletions and chromosome loss in gene-edited human primary T cells. These findings demonstrated that pre-selecting target sites favoring small insertions via guide RNA optimization coupled with CasPlus editing is a safer and more effective strategy to improve genome stability in T-cell engineering and other gene-editing applications.