Optical pooled screening (OPS) has emerged as a powerful technique for functional genomics, enabling researchers to link genetic perturbations with complex cellular morphological phenotypes at unprecedented scale. However, OPS data analysis presents challenges due to massive datasets, complex multi-modal integration requirements, and the absence of standardized frameworks. Here, we present Brieflow, a computational pipeline for end-to-end analysis of fixed-cell optical pooled screening data. We demonstrate Brieflow’s capabilities through reanalysis of a CRISPR-Cas9 screen encompassing 5,072 fitness-conferring genes, processing more than 70 million cells with multiple phenotypic markers. Our analysis reveals functional gene relationships that were missed in the original study, uncovering coherent biological insights related to mitochondrial function, mRNA processing, vesicular trafficking, and MYC transcriptional control, amongst others. The modular design and open-source implementation of Brieflow facilitates the integration of novel analytical components while ensuring computational reproducibility and improved performance for the use of high-content phenotypic screening in biological discovery.

The plant root microbiome is vital in plant health, nutrient uptake, and environmental resilience. To explore and harness this diversity, we present metagRoot, a specialized and enriched database focused on the protein families of the plant root microbiome. MetagRoot integrates metagenomic, metatranscriptomic, and reference genome-derived protein data to characterize 71,091 enriched protein families, each containing at least 100 sequences. These families are annotated with multiple sequence alignments, CRISPR elements, Hidden Markov Models, taxonomic and functional classifications, ecosystem and geolocation metadata, and predicted 3D structures using AlphaFold2. MetagRoot is a powerful tool for decoding the molecular landscape of root-associated microbial communities and advancing microbiome-informed agricultural practices by enriching protein family information with ecological and structural context. The database is available at https://pavlopoulos-lab.org/metagroot/ or https://www.metagroot.org

CRISPR-Cas9-based gene editing is a powerful approach to developing gene and cell therapies for several diseases. Engineering cell therapies requires accurate assessment of gene editing allelism because editing patterns can vary across cells leading to genotypic heterogeneity. This can hinder development of robust cell therapies. Droplet-based targeted single-cell DNA sequencing (scDNAseq) has been used to genotype targeted loci across thousands of cells enabling high-throughput assessment of gene editing efficiency. Here, we constructed a “ground truth” gene editing single-cell DNAseq atlas, along with an artifact-aware computational workflow called GUMM (Genotyping Using Mixture Models) to systematically infer single-cell allelism from these data. This resource was created by expanding CRISPR-Cas9-edited HL-60 clones that harbored distinct insertion-deletion (indel) profiles in CLEC12A and mixing them at pre-defined ratios to create artificial cocktails that mimic the potential editing diversity of a CRISPR-Cas9 experiment. This enabled assessment of technical artifacts that confound interpretation of allelism in the readouts of gene edited cells. GUMM was able to accurately genotype cells and infer the original clonal composition of the artificial cocktails even in the presence of artifacts.

Many cancer types upregulate expression of sialic acid-containing glycans. These oligosaccharides subsequently engage inhibitory Siglec receptors on immune cells, allowing cancer cells to evade immune surveillance. The genetic mechanisms by which this glycome remodeling occurs remain poorly defined. Understanding the ways that cancer cells change their cell surface glycosylation is critical for identification of biomarkers and targets for glycan-directed immunotherapy. In this study, we performed multiple gain-of-function CRISPR activation (CRISPRa) screens to broadly define genetic pathways that regulate expression of Siglec-binding glycans. We show that Siglec ligand expression is largely controlled through genetic competition between genes that catalyze α2-3 sialylation and GlcNAcylation of galactose residues. Perturbation of enzyme expression at this key biosynthetic node provides multiple “paths” by which cancers can acquire elevated expression of Siglec ligands. We further show that cancer glycome remodeling is aided by overexpression of novel “professional ligands” that facilitate Siglec-glycan binding. Notably, we also find that expression of the CD24 gene is genetically dispensable for cell-surface binding of the inhibitory receptor Siglec-10. Finally, by integrating our functional genetic model with clinical tumor genomic data, we identify the sulfotransferase enzyme GAL3ST4 as a potential novel driver of immune evasion in glioma cells. Taken together, this study provides a first-in-class genomic atlas to aid understanding of cancer-associated glycosylation and identifies immediately actionable targets for cancer immunotherapy.

Artemisia annua is renowned for producing artemisinin, a compound that revolutionized malaria treatment and holds therapeutic promise for other diseases, including cancer and diabetes. However, low natural yields of artemisinin remain a major bottleneck, necessitating a deeper understanding of the genetic and regulatory networks involved in its biosynthesis. Although several transcriptomic studies on A. annua exist, they are often limited in scope, and a comprehensive, tissue-resolved gene expression resource has been lacking. Here, we present the Artemisia Database (Artemisia-DB)—a high-resolution expression atlas constructed from an extensive integration of publicly available RNA-seq datasets. The database provides transcript- and gene-level abundance estimates across major tissues and includes functional annotations such as Gene Ontology (GO) terms, KEGG pathways, and InterPro domains. As a case study, we investigated the coexpression profile of HMGR (3-hydroxy-3-methylglutaryl- CoA reductase), a key enzyme in the mevalonate pathway and an early step in artemisinin biosynthesis. Coexpression analysis in leaf tissue revealed a subset of Auxin Response Factor (ARF) transcription factors strongly correlated to HMGR. This finding suggests a potential regulatory link between auxin signaling and artemisinin biosynthesis, providing new hypotheses for functional validation. Artemisia-DB is freely accessible at https://artemisia-db.com and offers an interactive interface for exploring expression data, functional annotations, transcription factors, CRISPR targets, and more. By combining high-quality transcriptome data with regulatory and functional insights, Artemisia-DB serves as a valuable resource for the plant research community and facilitates deeper investigation into the transcriptional dynamics and specialized metabolism of A. annua .

In recent decades, the idea to develop crop varieties which can handle environmental stressors has been taking form. Many effective genetic engineering tools have been developed, but many of those techniques are prone to ecological risks and face challenges from regulatory approvals and public perspectives. So this paper outlines a non-transgenic approach which battles transgenic constraints through a method called SmartNative Genome Editing, in which crop resilience is increased by using its native gene sequences. The main components of SmartNative Genome Editing include Cisgenic Regulatory Editing (CRE) and Epigenetic Priming-Based Trait Modulation (EPTM). Cisgenic regulatory editing (CRE) modifies the native regulatory patterns of plants using CRISPR-Cas9. Epigenetic Priming-Based Trait Modulation (EPTM) involves modification through epigenetic modifiers to mimic the actual environmental stress and simulate stress memory in plants, by which plants can easily respond faster when real conditions prevail. With both CRE and EPTM methods regulating gene expression without inserting foreign DNA will be a safer and more acknowledged pathway for crop improvement and have better environmental adaptability.

CRISPR-Cas systems have revolutionized modern biology. Most CRISPR-Cas systems in use for biotechnological applications derive from cultivated bacteria, but evidence suggests that environmental microbiomes harbor a large untapped diversity of these systems. Yet, our understanding of which environmental and biological factors drive the prevalence of CRISPR-Cas systems in the oceans remains limited. A search for CRISPR-Cas systems was conducted among 176 globally-distributed marine microbial metagenomes from the Malaspina expedition, which sampled both free-living and particle-attached microbiomes with emphasis on the deep ocean. We show that CRISPR-Cas systems are proportionally more abundant among microbiomes from the deep ocean than in the photic layers and among free-living microbes compared to those attached to particles, reflecting the higher concentrations of archaea and their viruses in these habitats. We identified 1,146 CRISPR- cas loci, some of which displayed unique loci architectures. From these loci, a total of 48 Cas9 proteins were identified, many of which are potentially novel. These discoveries expand the scope of CRISPR-Cas diversity and point at the deep-sea as a rich reservoir of these resources, which helps guide future bioprospecting efforts.

Abstract Sepsis is a heterogeneous clinical syndrome with a high mortality rate and personalised stratification strategies are proposed as essential to successful targeted therapeutics. Here, we characterise genetic variation that modulates MTOR , a critical regulator of metabolism and immune responses in sepsis. The effects are highly context specific, involving a regulatory element that affects MTOR expression in activated T cells with opposite direction of effect in neutrophils. The lead variant, rs4845987, significantly interacts with the known sepsis prognostic marker neutrophil-to-lymphocyte ratio, shows activity specific to sepsis endotype, and a pleiotropic effect on type 2 diabetes (T2D) risk. Using ex vivo models, we demonstrate that activated T cells promote immunosuppressive sepsis neutrophils through released cytokines, a process dampened by hypoxia and the mTOR inhibitor rapamycin. The G-allele of rs4845987, associated with decreased risk of T2D, is associated with reduced mTOR signaling in T cells and improved survival in sepsis patients due to pneumonia. We define a novel epigenetic mechanism that fine-tunes MTOR transcription and T cell activity via the variant-containing regulatory element, which exhibits an allelic effect upon vitamin C treatment. Our findings reveal how common genetic variation can interact with disease state/endotype to modulate immune cell-cell communication, providing a patient stratification strategy to inform more effective treatment of sepsis.

Antigenic variation is a sophisticated immune evasion strategy employed by many pathogens. Trypanosoma brucei expresses a single Variant-Surface-Glycoprotein (VSG) from a large genetic repertoire, which they periodically switch throughout an infection. Co-transcribed with the active- VSG within a specialised nuclear body are expression-site-associated-genes ( ESAGs ), involved in important host-parasite interactions, including protecting the parasite from human serum lytic effects, modulating the host’s innate immune response and uptake of essential nutrients. Despite expression within the same polycistron, there is a significant differential expression between ESAGs and VSGs (>140-fold), however, the regulatory mechanism has remained elusive for decades. Here, using a combination of genetic tools, super resolution microscopy, proteomics and transcriptomics analyses, we identified three novel proteins, which are recruited in a hierarchical manner, forming discreet sub-nuclear condensates that are developmentally regulated and negatively regulate ESAG transcripts. Among them, Expression-Site-Body-specific-protein-2 (ESB2) contains a nuclease domain that shares structural similarity to the endonuclease domain found in SMG6, a critical component of nonsense mediated decay in mammals. Mutation of key residues required for the nuclease activity impaired ESB2 localisation and function. Overall, our findings reveal a novel mechanism of post-transcriptional regulation and shed light on how specialised RNA decay can regulate expression of specific genes.

Many cell fate decisions in the developing neural tube are directed by cross-repressive transcription factor (TF) motifs that generate bistability, enforcing expression of one dominant TF. However, evidence of hybrid states, where cells co-express opposing fate determinants, challenges this model. We hypothesised that oscillatory expression enables co-existence of cross-repressive TFs within single cells, allowing hybrid states in bistable motifs. To test this, we focused on HES1 and HES5, oscillatory, cross-repressive TFs that regulate neural progenitor maintenance and are expressed in adjacent dorsoventral domains in the developing spinal cord. Using live-cell imaging of fluorescent reporters and computational modelling, we show that HES1 and HES5 co-express and oscillate in-phase within single cells. Differences in protein stability result in distinct free-running periodicity, but co-expression results in entrainment and phase-locking. Modulating cross-repression strength and/or abundance shifts the system towards bistability and dominance of a single TF oscillator. Consistent with this, we observe progressive separation of the HES expression domains in vivo, through a decrease in oscillatory co-expression. Our findings provide a mechanism for hybrid states to emerge in a developmental bistable motif.

Genetic screens in organoids hold tremendous promise for accelerating discoveries at the intersection of genomics and developmental biology. Embryoid bodies (EBs) are self-organizing multicellular structures that recapitulate aspects of early mammalian embryogenesis. We set out to perform a CRISPR screen perturbing all transcription factors (TFs) in murine EBs. Specifically, a library of TF-targeting guide RNAs (gRNAs) was used to generate mouse embryonic stem cells (mESCs) bearing single TF knockouts. Aggregates of these mESCs were induced to form mouse EBs, such that each resulting EB was ’mosaic’ with respect to the TF perturbations represented among its constituent cells. Upon performing single cell RNA-seq (scRNA-seq) on cells derived from mosaic EBs, we found many TF perturbations exhibiting large and seemingly significant effects on the likelihood that individual cells would adopt certain fates, suggesting roles for these TFs in lineage specification. However, to our surprise, these results were not reproducible across biological replicates. Upon further investigation, we discovered cellular bottlenecks during EB differentiation that dramatically reduce clonal complexity, curtailing statistical power and confounding interpretation of mosaic screens. Towards addressing this challenge, we developed a scalable protocol in which each individual EB is monoclonally derived from a single mESC and genetically barcoded. In a proof-of-concept experiment, we show how these monoclonal EBs enable us to better quantify the consequences of TF perturbations as well as ’inter-individual’ heterogeneity across EBs harboring the same genetic perturbation. Looking forward, monoclonal EBs and EB-derived organoids may be powerful tools not only for genetic screens, but also for modeling Mendelian disorders, as their underlying genetic lesions are overwhelmingly constitutional ( i.e. present in all somatic cells), yet give rise to phenotypes with incomplete penetrance and variable expressivity.

Ruminant gut microbial communities profoundly influence host health and environmental impacts, yet their viral components remain poorly characterised across developmental transitions. Here, we analysed the dairy cow gut virome across four life stages—calf, heifer, dry adult, and lactating adult. Using hybrid sequencing, we assembled 30,321 viral operational taxonomic units, including 1,338 complete genomes representing mostly novel lineages. Virome composition shifted dramatically with life stage, transitioning from low-diversity, temperate-dominated communities in calves to high-diversity, lytic-dominated communities in adults. Virome transitions paralleled but showed distinct dynamics from bacterial community development, with viral and bacterial diversity negatively correlated during drying-off. Dry cows exhibited elevated viral loads relative to their bacterial hosts. We identified 26 viral sequences targeting the methanogen Methanobrevibacter , absent in calves but present in adults. These findings reveal the dynamic nature of ruminant gut viral communities and highlight phages’ potential regulatory role during critical life transitions.

The noncanonical translation initiation factor eIF2A plays critical roles in diverse cellular processes, including the integrated stress response, neurodegeneration and tumorigenesis. However, the precise molecular mechanism underlying eIF2A’s function remains poorly understood. Here, we exploit a TurboID-based proximity labeling combined with mass spectrometry to systematically map the interactome of eIF2A during homeostasis and stress. Combining polysome gradients with TurboID, we zoom into the interactions of eIF2A with the 40S small ribosomal subunit and map the eIF2A binding site close to the mRNA entry channel. We identify a network of interactors that link eIF2A to ribosome-associated quality control, including its strong interaction with G3BP1-USP10 complexes as well as RPS2 and RPS3. In the absence of eIF2A, RPS2 and RPS3 ubiquitination is diminished specifically upon ribosome stalling. 40S-specific footprinting in eIF2A knockout cells shows minimal changes in 5’UTR occupancy, consistent with a limited role for eIF2A in translation initiation. Using dynamic SILAC mass spectrometry, we characterize the novel function of eIF2A in ribosome-associated quality control and show that eIF2A antagonizes USP10-dependent rescue of 40S ribosomes, resulting in altered turnover of 40S subunits upon cellular stress. Collectively, our study identifies a previously unknown link between eIF2A and ribosome-associated quality control, implies that eIF2A promotes translation fidelity by tuning 40S ribosome rescue under stress and warrants further investigations into the role of ribosome-associated quality control in tumorigenesis.

Antiretroviral therapy (ART) suppresses HIV replication but fails to eliminate the virus due to the persistence of a transcriptionally silent reservoir, which remains the primary barrier to a cure. HIV latency is maintained through chromatin-mediated repression, making epigenetic regulators attractive therapeutic targets. To identify new modulators of latency, we screened a focused library of 84 chromatin-targeting small molecules. This screen identified BAY-299, a bromodomain inhibitor selective for TAF1 and BRD1, as a latency-modulating compound. BAY-299 reactivated HIV expression and enhanced the efficacy of established latency-reversing agents (LRAs), including vorinostat, prostratin, and iBET-151, in cell line models. CRISPR/Cas9-mediated knockout experiments demonstrated that TAF1, but not BRD1, is essential for maintaining HIV latency and that TAF1 depletion selectively increases HIV transcription with minimal effects on host gene expression. Dual knockout of TAF1 and Tat revealed that the reactivation effect is partially Tat dependent. CUT&RUN analysis further showed that TAF1 depletion increases RNA Polymerase II occupancy across the HIV gene body, suggesting enhanced transcriptional elongation. These findings identify TAF1 as a novel regulator of HIV latency and demonstrate the utility of targeted chemical screening to uncover therapeutic vulnerabilities within the latent reservoir. Importance HIV remains incurable due to the persistence of a transcriptionally silent reservoir in infected cells that is not eliminated by antiretroviral therapy. This transcriptionally silent state, known as latency, is controlled by host cell factors that regulate access to the viral genome. In this study, we identified the host protein TAF1 as a key regulator that maintains HIV in a latent state. Using both genetic and chemical approaches, we demonstrated that reducing TAF1 levels selectively increases HIV gene expression without broadly disrupting host gene transcription. These findings highlight a previously unrecognized mechanism of HIV latency control and identify TAF1 as a potential therapeutic target. Understanding how host chromatin regulators contribute to latency is essential for developing strategies that aim to eliminate the persistent HIV reservoir.

Creating hypomorphic mutations are crucial to study gene function in vivo , especially when null mutations result in (embryonic) lethality. This is especially the case for enzymes involved in glycosylation that, when mutated in human patients, are causing the disease congenital disorders of glycosylation (CDG). To resemble the patient conditions, it would be ideal to acutely modulate the proteins in question to directly interfere with protein levels of such essential enzymes. These methods offer to establish pathogenic enzyme levels resembling net enzyme activity reported from patients suffering from CDG, with Phosphomannomutase 2 - CDG (PMM2-CDG) as the most common form. We established an auxin-inducible acute protein knockdown system for the use in the teleost fish medaka ( Oryzias latipes ) by combining an improved degron (AID2) technology with a mAID-nanobody targeting endogenously GFP-tagged Pmm2 protein. We generated a fishline expressing a functional Pmm2-GFP fusion protein, by single copy integration of GFP into the pmm2 locus. Upon induction, the degron system efficiently reduced Pmm2-GFP levels and enzyme activity, recapitulating the activity level of the hypomorphic mutations associated with PMM2-CDG in patients. This broadly applicable approach enables the investigation of CDG disease mechanisms during early embryonic development through reduction of protein abundance mimicking hypomorphic mutations and thus substantially expands the range of the genetic toolbox. Summary Statement The combination of TIR1F74G and mAID-GFP-nanobody enables efficient acute knockdown of endogenously GFP-tagged proteins in medaka. This approach successfully reduced Pmm2 enzyme activity to pathological levels as seen in PMM2-CDG patients.

Genomic alterations driving tumorigenesis in sinonasal malignancies remain largely unexplored. Here, we perform an in vivo loss-of-function screen using a pooled custom single-guide library delivered to the sinonasal cavity by adeno-associated virus vector to identify cancer driver genes across diverse sinonasal malignancies. This approach yielded sinonasal malignancies with diverse histologies, including sinonasal squamous cell carcinoma, adenocarcinoma, poorly differentiated sinonasal carcinoma, and sinonasal neuroendocrine tumors characteristic of olfactory neuroblastoma. Surprisingly, rather than observing distinct sgRNA profiles across sinonasal tumor subtypes, common recurrent mutations were identified in Nf1 (79%), Rasa1 (74%), and Trp53 (68%) across malignancies with distinct histologies. Utilizing an orthogonal approach, we confirmed that Nf1/Trp53 were required for sinonasal tumorigenesis. Given that loss-of-function in NF1 and RASA1 may lead to increased Ras activity and downstream MEK signaling, we tested small molecule targeting of the RAS-MAPK pathway in sinonasal malignancies. Indeed, both tumor cell lines derived from our loss-of-function approach as well as from human sinonasal malignancies displayed significant sensitivity to MEK inhibition in standard in vitro culture and organoid models. These findings demonstrate that loss of NF1 and RASA1-mediated Ras-GAP activity leads to Ras activation and downstream MEK signaling which is a potential common target throughout major sinonasal tumor subtypes.

The fatty acid elongase1 ( FAE1 ) genes of tetraploid Brassica juncea are the key determinant of high erucic acid (EA, C22:1) accumulation in its seed oil. While our previous work demonstrated near-zero EA content in mustard oil via CRISPR/Cas9 knockout of the two homeoalleles, BjFAE1.1 and BjFAE1.2 ; the contributory function of each isozymes towards EA biosynthesis remains elusive. This study investigated the heterologous expression of BjFAE1.1 and BjFAE1.2 from high EA B. juncea cultivar JD6 in two metabolically distinct eukaryotic microbial hosts: the green microalga Chlamydomonas reinhardtii and the budding yeast Saccharomyces cerevisiae . Despite confirmed protein expression, neither BjFAE1 isozyme produced detectable C20:1 or C22:1 very-long-chain fatty acids (VLCFAs) in transgenic lines of C. reinhardtii . In contrast, expression in S. cerevisiae resulted in significant de novo biosynthesis of VLCFAs, C20:1 (∼9-11%) and C22:1 (∼17-19%), confirming their enzymatic activity as functional β-ketoacyl-CoA synthase. Substrate feeding experiments in yeast further validated their capability to elongate oleoyl-CoA (C18:1-CoA) to erucoyl-CoA (C22:1-CoA) via eicosenoyl-CoA (C20:1-CoA), with BjFAE1.1 showing slightly higher activity, as indicated by the enhanced VLCFAs accumulation. These findings highlight the critical influence of the heterologous host’s cellular environment on the enzyme functionality of plant genes involved in lipid metabolism, underscoring challenges for VLCFA production in microalgal platform.

Papaya ( Carica papaya L.) is an economically important tropical crop that produces papain and highly nutritious fruit, which are used in the grocery, cosmetic, pharmaceutical, and food processing industries. However, various destructive pathogens severely threaten its production. Furthermore, limited natural genetic variation restricts breeding efforts for crop improvement. Therefore, we turned to gene editing as a tool to address these problems. We utilized two CRISPR systems (Cas9 and Cas12a) and two papaya genes, CpPDS (phytoene desaturase) and CpMLO6 (Mildew Locus O 6), to establish efficient genome editing systems of papaya. The systems were delivered by an optimized protocol of Agrobacterium -mediated transformation (AMT) of embryogenic callus suspension cultures derived from hypocotyls. Accordingly, we transformed papaya with five plasmid constructs, each of which expressed one or two guide RNAs (gRNAs) for gene editing using either Cas9 or Cas12a. All except two T0 transgenic plants tested produced mutations with the majority containing indels of over 90%. Furthermore, successful mutation of the CpPDS gene using both Cas9 and Cas12a produced albino phenotypes as expected for disrupting a gene for carotenoid biosynthesis. Successful mutagenesis was achieved with seven out of eight gRNAs. Homozygous and/or biallelic mutants were generated from transformation using all five constructs, suggesting the feasibility of obtaining transgene-free homozygous segregating mutants by selfing in the second generation. Taken together, a robust and reliable papaya genome editing system was established, which enables genetic modification in various genomic environments to meet the diverse needs of basic scientific research and tropical crop improvement.

Plants constantly monitor their environment to adapt to potential threats to their health and fitness. This involves cell-surface receptors that can detect conserved microbe-associated molecular patterns (MAMPs) or endogenous immunogenic signals, initiating signaling pathways to induce broad-spectrum disease resistance, known as pattern-triggered immunity (PTI). In Arabidopsis thaliana , the leucine-rich repeat receptor kinase (LRR-RK) MIK2 is an exceptionally versatile receptor involved in the perception of the vast family of Brassicales-specific endogenous SCOOP peptides as well as potential MAMPs derived from Fusarium and related fungi. Although only plant species belonging to the order of Brassicales encode genes for SCOOP peptides and show SCOOP-responsiveness, the Fusarium -derived elicitor fraction also induces PTI responses in plants from other lineages. In this study, we demonstrate that Fusarium elicitor-responsiveness and proteins belonging to the MIK2-clade are widely conserved among seed plants. We identified a MIK2-clade protein from tomato, which shares properties of At MIK2 in the perception of the Fusarium elicitor but not of SCOOP peptides. Tomato mutants lacking the receptor show compromised PTI responses to the fungal elicitor and enhanced susceptibility to infection by Fusarium oxysporum . Our data provide insights into the evolutionary trajectory of MIK2 as a multifunctional receptor involved in plant immunity.

Abstract Recent single-cell CRISPR screening experiments have combined the advances of genetic editing and single-cell technologies, leading to transcriptome-scale readouts of responses to perturbations at single-cell resolution. An outstanding question is how to efficiently identify heterogeneous effects of perturbations using these technologies. Here we present CausalPerturb, which leverages AI tools and causal analysis to dissect the heterogeneous landscape of perturbation effects. CausalPerturb disentangles transcriptome changes introduced by perturbations from those reflecting inherent cell-state variations. It provides nonparametric inferences of perturbation effects, enabling a range of downstream tasks including genetic interaction analysis, perturbation clustering and prioritization. We evaluated CausalPerturb through simulation studies and real datasets, and demonstrated its competence in characterizing latent confounding factors and discerning heterogeneous perturbation effects. The application of CausalPerturb unraveled novel genetic interactions between erythroid differentiation drivers. In particular, it pinpointed the role of the synergistic interaction between CBL and CNN1 in the S phase.

Abstract Insights from urochordates/tunicates can instruct on the evolutionary origins of key cell types or gene regulatory mechanisms such as for the ‘new head’ sensory placodes and neural crest. Taking advantage of their invariant lineage with reproducible binary cell fate switches, we decipher in Ciona intestinalis the unanswered question of how highly conserved and ongoing FGF/MAPK/ERK signalling gives rise to co-existing nuclear Ets activation and repression states to finely tune the neural fate and its diversification. Genetic interference shows that Erf and Elk repressors play successive roles at different transcriptional targets. We propose an Ets site occupancy model where activators and repressors compete to produce consecutive and opposite winning switches in adjacent territories. Such Ets factor network is relevant beyond the ascidian neuroectodermal lineage to produce palp placodal and neural plate progenitors. It may explain Ets factor effects in many metazoans including Elk in vertebrate neural crest and appeal to stem cell and cancer research.

Abstract Background. Metastatic breast cancer (MBC) remains a major clinical challenge, particularly in estrogen receptor α (ERα)-positive patients who develop resistance to endocrine therapy (ET). While hotspot mutations such as Y537S in the ligand-binding domain (LBD) are well-characterized drivers of resistance, other ERα variants remain poorly studied. Understanding the molecular mechanisms underlying resistance in these variants is crucial for identifying novel therapeutic strategies. Here, we investigated the functional role of the L370F and E471D ERα variants, which are spatially close in the ERα structure. Methods. Stable overexpressing HEK293 cells and CRISPR/CAS9 engineered MCF-7 cells were generated and treated with 17β-estradiol (E2), fulvestrant (Ful) and all-trans retinoic acid (ATRA) to measure ERα stability, transcriptional activity and gene expression analyses using different cellular assays and RNASeq techniques. Direct in vitro measurement of ligand binding affinity to ERα were performed using the purified full-length wild type (wt) as well as L370F and Y537S ERα. In silico structural simulations were also performed to predict the structure of the mutated L370F ERα. Senescent analyses of MCF-7 and Y537S MCF-7 cells were performed using direct measurement β-galactosidase activity in vitro and in cell lines. Results The L370F variant conferred resistance to Ful in terms of in vitro ERα binding, ERα transcriptional activity, receptor degradation and cell proliferation by modifying the folding of the receptor structure. Furthermore, L370F-expressing cells exhibited a hyperactive response to low doses of E2 and basally upregulated late estrogen responsive genes. Additionally, we found that both L370F and Y537S ERα variants displayed increased RARα expression, rendering them highly sensitive to ATRA. Notably, ATRA killed L370F-expressing cells and induced senescence in Y537S-expressing cells, highlighting mutation-specific responses. Conclusions Our findings expand the understanding of ERα mutations beyond known hotspots, identifying L370F as a novel mutation contributing to ET resistance and further indicate the necessity to characterize all the less-studied ERα variants found in MBC. Furthermore, we demonstrate that ATRA selectively targets MBC cells harboring L370F and Y537S mutations, suggesting its potential as a mutation-specific therapeutic agent. These results support further investigation of ATRA in clinical settings to improve treatment strategies for ERα-mutant MBC.

ABSTRACT TP53 mutations confer treatment resistance across multiple cancers. Mechanisms of therapy resistance, beyond affecting transactivation of BCL-2 family genes, remain a mystery. Here, we report that TP53 mutated AML, triple negative breast cancer, and colorectal cancer escape therapy-induced apoptosis due to inability to activate caspase-3/7, despite having normal mitochondrial outer membrane permeabilization (MOMP) induction. To identify post-MOMP determinants of therapy resistance in TP53 mutated AML, we applied a multiomics approach – whole-genome CRISPR screen, bulk/single-cell RNAseq, and high-throughput drug screen. BIRC5 , encoding survivin, was selectively upregulated in paired hematopoietic stem/multipotent progenitor cells from TP53 mutant AML patients, with further enrichment after venetoclax-azacitidine (VenAza) relapse. Critically, BIRC5 was also upregulated in 17 of 26 TP53 mutant TCGA cancers. Genetic ablation of BIRC5 resensitized TP53 mutated AML to standard therapy by restoring caspase activation, validating therapeutic relevance. Importantly, targeting IAPs and survivin using clinically relevant inhibitors overcame VenAza resistance of TP53 mutant tumors in vivo , achieving sustained AML suppression. Combination with survivin inhibitors also overcame chemotherapy resistance in TP53 deficient solid cancers. Together, we discovered that wild-type TP53 is required in post-MOMP signaling and that BIRC5 dependency is an effective therapeutic target for poor prognosis, TP53 mutated cancers.

Immune matching and rejection pose major hurdles in tissue transplantation. Here, we profile HLA-A , HLA-B , and HLA-C alleles in 3,496 Lithuanian donors genotyped at three-field resolution. The five most frequent alleles constitute 74.6% of HLA-A , 43.2% of HLA-B , and 59.2% of HLA-C , with HLA-A*02:01:01, HLA-B*07:02:01, and HLA-C*07:02:01 being the most common. Lithuanian allele frequencies closely resemble those of populations with pre-Neolithic hunter-gatherer ancestry, such as European-American and British groups. We identified 153 double homozygotes and 51 triple homozygotes for HLA-A , HLA-B , and HLA-C . Compatibility modeling showed triple homozygous profiles match 60.5% of Lithuanians (33.3% for double homozygotes), 13.4% of British population, and 7.4% of European-Americans. CRISPR-Cas9 guide RNA design yielded 54 candidates predicted to disrupt HLA-A or HLA-B , while preserving HLA-C , producing edited profiles matching over 98.1% of Lithuanians, 95.8% of European-Americans, and 95.6% of British population. Finally, we established 16 fibroblast lines from double and triple homozygotes, offering a resource for immune-compatibility studies and regenerative medicine applications.

RNA splicing is fundamental to cellular function, yet systematic investigation of its complex regulation has been limited by existing methods. Here, we present SPLiCR-seq ( SPL icing regulator identification through CR ISPR screening), a high-throughput CRISPR screening platform that enables direct measurement of RNA splicing outcomes for pooled genetic perturbations, overcoming limitations of traditional fluorescence-based approaches. Applying SPLiCR-seq to investigate XBP1 splicing during the unfolded protein response (UPR), we conducted targeted and genome-wide screens across diverse cellular contexts, revealing both common and cell-type specific regulators. Notably, we identified GADD34 ( PPP1R15A ) as a novel modulator of IRE1-XBP1 signaling, demonstrating that it directly interacts with IRE1 and functions independently of its canonical role in eIF2α dephosphorylation. Pharmacological inhibition of GADD34 using Sephin1 effectively suppressed XBP1 splicing and alleviated CAR-T cell exhaustion in an ex vivo model, leading to enhanced tumor-killing capacity across multiple cancer models. This work not only establishes a powerful new tool for systematically studying RNA splicing regulation but also uncovers a promising therapeutic strategy for improving CAR-T cell immunotherapy through modulation of the IRE1-XBP1 pathway.