ABSTRACT In public health emergencies or in resource-constrained settings, laboratory-based diagnostic methods, such as RT-qPCR, need to be complemented with accurate, rapid, and accessible approaches to increase testing capacity, as this will translate into better outcomes in disease prevention and management. Here, we develop an original nucleic acid detection platform by leveraging the CRISPR-Cas9 and lateral flow immunochromatography technologies. In combination with an isothermal amplification that runs with a biotinylated primer, the system exploits the interaction between the CRISPR-Cas9 R-loop formed upon targeting a specific nucleic acid and a fluorescein-labelled probe to generate a visual readout on a lateral flow device. Our method enables rapid, sensitive detection of nucleic acids, achieving a limit of 1-10 copies/μL in 1 h at low temperature. We validated the efficacy of the method using clinical samples of patients infected with SARS-CoV-2. Compared to other assays, it operates with more accessible molecular elements and showcases a robust signal-to-noise ratio. Moreover, multiplexed detection was demonstrated using primers labeled with biotin and digoxigenin, achieving simultaneous identification of target genes on lateral flow devices with two test lines. We successfully detected SARS-CoV-2 and Influenza A (H1N1) in spiked samples, highlighting the potential of the method for multiplexed diagnostics of respiratory viruses. All in all, this represents a versatile and manageable platform for point-of-care testing, thereby supporting better patient outcomes and enhanced pandemic preparedness.

Abstract Backgrounds and Aim: Colorectal cancer (CRC) pathogenesis is correlated with dysregulation of tight junction. This study aimed to investigate the molecular mechanism by which trimethylamine N-oxide (TMAO) alters the expression of tight junction proteins in a colorectal cancer (CRC) cell line. Material and Method: The study utilized the CRISPR/Cas13 system for targeted knock down of HULC in Caco-2 cells, followed by treatment with trimethylamine N-Oxide (TMAO). Tight junction components, including ZO-1, Claudin-1, and Occludin, were analyzed using real-time quantitative polymerase chain reaction (RT-qPCR). To investigate the role of the P38MAPK pathway, the specific inhibitor SB203580 was used in cells treated with TMAO to comprehensively assess tight junction regulation. Statistical analysis was performed using one-way ANOVA to compare the mean ± SD between different groups, followed by paired comparisons using the t-test. Results: Cells treated with TMAO showed a significant upregulation of the oncogenic long non-coding RNA (lncRNA) HULC (Highly Upregulated in Liver Cancer), , accompanied by increased expression of p38 MAPK. Interestingly, a significant downregulation of ZO-1 and Claudin-1 was observed as a result of TMAO treatment, which was modulated by the HULC/p38 MAPK axis. However, Occludin expression was also reduced by TMAO, but it remained unaffected by the HULC/p38 MAPK pathway. Conclusion: This study revealed a novel TMAO/HULC/p38 MAPK axis involved in the regulation of tight junctions in a colorectal cancer cell line model. TMAO treatment significantly reduced the expression of ZO-1, Claudin-1, and Occludin. Further in vivo research is strongly recommended to clarify the impact of TMAO on the integrity of colorectal cancer cells.

The second messenger bis -(3′→5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) governs adaptive responses in the opportunistic pathogen Pseudomonas aeruginosa , including biofilm formation and the transition from acute to chronic infections. Understanding the intricate c-di-GMP signaling network remains challenging due to the overlapping activities of numerous diguanylate cyclases (DGCs). In this study, we employed a CRISPR-based multiplex genome-editing tool to disrupt all 32 GGDEF domain-containing proteins (GCPs) implicated in c-di-GMP signaling in P . aeruginosa UCBPP-PA14. Phenotypic and physiological analyses revealed that the resulting mutant was unable to form biofilms and had attenuated virulence. Residual c-di-GMP levels were still detected despite the extensive GCP disruption, underscoring the robustness of this regulatory network. Taken together, these findings provide insights into the complex c-di-GMP metabolism and showcase the importance of functional overlapping in bacterial signaling. Moreover, our design overcomes the native redundancy in c-di-GMP synthesis, providing a framework to dissect individual DGC functions and paving the way for targeted strategies to address bacterial adaptation and pathogenesis.

Cultivated oat ( Avena sativa ) is an emerging cereal for healthy lives owing to its unique characteristics, such as high β-glucan and oil content, distinctive fatty acid composition, and gluten-free nature. The recent unravelling of the 12.5 Gb hexaploid oat genome underlined breeding barriers caused by ancestral translocations and inversions, leading to recombination suppression and pseudo-linkage further hindering conventional trait introgression. Over the past decade, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system has been extensively used for crop improvement and functional genomics in all other cereals except oats. Its large repetitive genome with three sub-genomes, lack of efficient transformation, recalcitrant nature, and complex molecular screening due to gene redundancy have been major obstacles to gene editing success in oat. We report the first successful CRISPR-Cas9-based gene editing in oat in three genes — AsTLP8, AsVRN3 and AsVRN3D with gene-editing efficiency of up to 41.1%. The gene-edited plants for all the genes carried deletions and/or one base insertion. Further analysis of VRN3 T 1 and T 2 mutants revealed bent leaves in heterozygous knockouts (AACCdD), while an extended vegetative growth phase was seen in the T 1 homozygous and biallelic mutants (aaccdd), accentuating the important role of VRN3 in oat development. We are confident that this highly efficient oat gene editing system will pave the way for a deeper molecular understanding of this healthy cereal, deciphering oat’s functional genomics, and creating genetic diversity at the cold spots of recombination in oat.

ABSTRACT Arthropods have an incredible diversity of limbs that are modified for walking, chewing, cleaning, mating, grasping, sensing, and more. Understanding the relationships and evolutionary histories of different limbs is a central task, but their sheer diversity makes this a daunting if not impossible task using morphology alone. Here, the in situ expression patterns and CRISPR-Cas9 phenotypes for the five best-studied leg-patterning genes – Distal-less , Sp6-9 , dachshund , extradenticle , and homothorax – are described for all limbs of the crustacean Parhyale . Crustaceans are well-suited for this task because their limbs are more diverse than those of other arthropods, and each individual possesses a wide range of limb types that are relevant to many other arthropods, living and extinct. These results will a) provide a template for understanding the genetic basis of limb construction in arthropods more generally based on the strong phenotypes that can be obtained with CRISPR-Cas9, and b) contribute to our understanding of the evolution and affinities of highly modified legs like mouthparts and genitalia using molecular methods to complement previous morphological and embryological approaches.

Motivation Understanding the factors involved in DNA double-strand break (DSB) repair is crucial for the development of targeted anti-cancer therapies, yet the roles of many genes remain unclear. Recent studies show that perturbations of certain genes can alter the distribution of sequence-specific mutations left behind after DSB repair. This suggests that genome-wide screening could reveal novel DSB repair factors by identifying genes whose perturbation causes the mutational distribution spectra observed at a given DSB site to deviate significantly from the wild-type. However, designing proper controls for a genome-wide perturbation screen could be challenging. We explore the idea that a genome-wide screen might allow us to forgo the use of traditional non-targeting controls by reframing the analysis as an outlier detection problem, assuming that most genes have minimal influence on DSB repair. Results We propose MUSICiAn (Mutational Signature Catalogue Analysis), a compositional data analysis method that ranks gene perturbation-specific mutational spectra without controls by measuring deviations from the central tendency in the distributions of all spectra. We show that MUSICiAn can effectively estimate pseudo-controls for the existing Repair-seq dataset, screening 476 genes and 60 non-targeting controls. We further apply MUSICiAn to a genome-wide dataset profiling mutational outcomes induced by CRISPR-Cas9 at three target sites across cells with individual perturbations of 18,406 genes. MUSICiAn successfully recovers known genes, highlights the spliceosome as a lesser-appreciated player in DSB repair, and reveals candidates for further investigation. Availability github.com/joanagoncalveslab/MUSICiAn .

The maize smut fungi Ustilago maydis and Sporisorium reilianum are closely related and have similar genomes in terms of size and synteny. While U. maydis induces tumors locally at sites of infection, S. reilianum systemically colonizes the host and causes symptoms in the inflorescences. To investigate the genetic basis of these differences, an interspecific recombinant hybrid (rUSH) with the mating type system of S. reilianum was generated. rUSH exhibited extensive in-planta proliferation, showing a S. reilianum -like phenotype at all developmental stages except teliospore formation. Transcriptome profiling revealed that expression of pathogenicity-related effector gene orthologs was induced in rUSH, but not in a wild-type hybrid control. Multiple transcriptome comparisons identified 253 differentially expressed one-to-one effector orthologs with distinct regulatory patterns, including cis-, trans-, and rUSH-specific regulation. Functional analysis via CRISPR/Cas9 mutagenesis uncovered three novel virulence factors among the rUSH-specific regulated effectors. Ultimately, rUSH facilitated to identify the transcription factor UmHdp2 as key regulator of U. maydis- induced tumorigenesis. Together, these findings highlight the utility of a recombinant, interspecific hybrid in unraveling the molecular mechanisms underlying pathogenic differences in closely related fungal pathogens.

CRISPR base editors are crucial for precise genome manipulation. Existing APOBEC-based cytosine base editors (CBEs), while powerful, exhibit indels and sequence context limitations, where TC-context preferences restrict effective editing of CC and GC motifs. To address these challenges, we evaluated various tRNA adenine deaminase (TadA)-derived CBEs, ultimately engineering zTadCBE that demonstrates high editing efficiency, minimized off-target effects, and an expanded targeting range. Our approach integrates beneficial mutations from TadA-based adenine base editors (ABEs) with SpRYCas9n-enhanced protospacer-adjacent motif (PAM) compatibility. Additionally, we engineered expanded-window zTadCBE variants, zTadCBE-ex1 and zTadCBE-ex2, to target wider nucleotide ranges, further increasing the versatility of this tool. To demonstrate the utility of zTadCBE variants in the functional assessment of genetic mutants, we generated a model for CDH23-associated hearing loss to validate the pathogenicity of a patient-specific variant in zebrafish. Furthermore, we induced a premature stop codon in the mediator complex gene med12 to inactivate its function using a CRISPR-STOP strategy and recapitulated patient-specific phenotypes in the founding (F0) generation. zTadCBE variants thus offer a robust set of CBEs for precise and efficient C-to-T editing in zebrafish, promising to advance the rapid functional assessment of genetic variants in vivo.

Examining early-branching animal phyla can help reconstructing the evolutionary origin of animal immune cells. Here we characterized the immune related cell program in the sea anemone Nematostella vectensis, a model organism representing the phylum Cnidaria that diverged ~600 million years ago from the rest of animals. By using a Nematostella transgenic reporter line expressing mCherry under the RLRb antiviral promoter we show that cells fluorescent upon stimulation with the viral mimic poly(I:C) are morphologically and transcriptomically distinct. These cells exhibit upregulation of immune effector and regulator genes. Cellular assays revealed an increase in phagocytic activity upon poly(I:C) treatment in this cell population. Lastly, we used a combination of immunofluorescent staining followed by FACS and bulk RNA sequencing, accompanied by single-cell transcriptomic analysis, to reveal the gene regulatory programs associated with immune cells in Nematostella. Comparing the sea anemone immune expression profile with gene expression of stony corals treated with the immunostimulant 2′3′-Cyclic GMP-AMP revealed remarkable similarity, suggesting that the immune response to viral challenge is conserved across the cnidarian class Hexacorallia. Altogether, this research uncovers the characteristics of a novel cnidarian immune cell type involved in antiviral immunity and offers insights into the evolutionary history of the innate immune system.

Africa, home to 1.4 billion people and the highest genetic diversity globally, harbors unique genetic variants crucial for understanding complex diseases like neurodegenerative disorders. However, African populations remain underrepresented in induced pluripotent stem cell (iPSC) collections, limiting the exploration of population-specific disease mechanisms and therapeutic discoveries. To address this gap, we established an open-access African Somatic and Stem Cell Bank. In this initial phase, we generated 10 rigorously characterized iPSC lines from fibroblasts representing five Nigerian ethnic groups and both sexes. These lines underwent extensive profiling for pluripotency, genetic stability, differentiation potential, and Alzheimer's disease and Parkinson's disease risk variants. CRISPR/Cas9 technology was used to introduce frontotemporal dementia-associated MAPT mutations (P301L and R406W). This collection offers a renewable, genetically diverse resource to investigate disease pathogenicity in African populations, facilitating breakthroughs in neurodegenerative research, drug discovery, and regenerative medicine.

Microproteins, short functional peptides encoded by small genes, are emerging as critical regulators of cellular processes, yet their roles in mitochondrial function and neurodegeneration remain underexplored. In this study, we identify NCBP2-AS2 as an evolutionarily conserved mitochondrial microprotein with significant roles in energy metabolism and neurogenesis. Using a combination of cellular and molecular approaches, including CRISPR/Cas9 knockout models, stoichiometric co- immunoprecipitation, and advanced imaging techniques, we demonstrate that NCBP2-AS2 localizes to the inner mitochondrial space and interacts with translocase of the inner membrane (TIM) chaperones. These interactions suggest a role in ATPase subunit transport, supported by the observed reductions in ATPase subunit levels and impaired glucose metabolism in NCBP2-AS2-deficient cells. In zebrafish, NCBP2-AS2 knockout led to increased astroglial proliferation, microglial abundance, and enhanced neurogenesis, particularly under amyloid pathology. Notably, we show that NCBP2-AS2 expression is consistently downregulated in human Alzheimer’s disease brains and zebrafish amyloidosis models, suggesting a conserved role in neurodegenerative pathology. These findings reveal a novel link between mitochondrial protein transport, energy metabolism, and neural regeneration, positioning NCBP2-AS2 as a potential therapeutic target for mitigating mitochondrial dysfunction and promoting neurogenesis in neurodegenerative diseases such as Alzheimer’s disease.

ABSTRACT Fibrosis and persistent inflammation are interconnected processes that inhibit axon regeneration in the mammalian central nervous system (CNS). In zebrafish, by contrast, fibroblast-derived extracellular matrix deposition and inflammation facilitate regeneration. However, the regulatory cross-talk between fibroblasts and the innate immune system in the regenerating CNS is not understood. Here, we show that zebrafish fibroblasts possess a dual role in inducing and subsequently resolving inflammation, which are both essential for regeneration. We identify a transient, injury-specific cthrc1a + fibroblast state with an inflammation-associated, less differentiated, and non-fibrotic profile. Induction of this fibroblast state precedes and contributes to the initiation of the inflammatory response. At the peak of neutrophil influx, cthrc1a + fibroblasts coordinate the resolution of inflammation. Disruption of these inflammation dynamics inhibits axon regeneration and alters the mechano-structural properties of the lesion environment. This establishes the biphasic inflammation control by dedifferentiated fibroblasts as a pivotal mechanism for CNS regeneration. ONE SENTENCE SUMMARY Dedifferentiated fibroblasts sequentially induce and resolve neutrophil-driven inflammation through cytokine release to facilitate axon regeneration after spinal cord injury in zebrafish. HIGHLIGHTS Time-resolved single-cell transcriptomics of zebrafish spinal cord regeneration. Spinal cord injury induces fibroblast dedifferentiation. Dedifferentiated fibroblasts sequentially induce and resolve inflammation. Dysregulation of inflammation dynamics alters mechano-structural tissue properties.

Small noncoding RNAs (smRNAs) play critical roles in regulating various cellular processes, including development, stress response, and disease pathogenesis. However, functional characterization of smRNAs remains limited by the scale and simplicity of phenotypic readouts. Recently, single-cell perturbation screening methods, which link CRISPR-mediated genetic perturbations to rich transcriptomic profiling, have emerged as foundational and scalable approaches for understanding gene functions, mapping regulatory networks, and revealing genetic interactions. However, a comparable approach for probing the regulatory consequences of smRNA perturbations is lacking. Here, we present Decoy-seq as an extension of this approach for high-content, single-cell perturbation screening of smRNAs. This method leverages U6-driven tough decoys (TuD), which form stable duplexes with their target smRNAs, for inhibition in the cell. Lentiviral-encoded TuDs are compatible with conventional single-cell RNA-sequencing (scRNA-seq) technologies, allowing joint identification of the smRNA perturbation in each cell and its associated transcriptomic profile. We applied Decoy-seq to 336 microRNAs (miRNAs) and 196 tRNA-derived fragments (tRFs) in a human breast cancer cell line, demonstrating its ability to uncover complex regulatory pathways and novel functions of these smRNAs. Notably, we show that tRFs influence mRNA polyadenylation and regulate key cancer-associated processes, such as cell cycle progression and proliferation. Therefore, Decoy-seq provides a powerful framework for exploring the functional roles of smRNAs in normal physiology and disease, and holds promise for accelerating future discoveries.

Latent EBV infection is causally associated with various B-cell malignancies, while periodic lytic-cycle replication is essential for sustaining viral progeny. Lytic cycle induction represents a promising therapeutic strategy for EBV-associated neoplasms. Therefore, uncovering the mechanisms that regulate EBV lytic-cycle reactivation is pivotal for understanding viral pathogenesis and advancing novel therapies. Our genome-wide transcriptomic analysis reveals that E2F1 expression is transcriptionally activated during EBV latent infection in B-lymphocytes but significantly suppressed during lytic cycle reactivation. While ectopic E2F1 expression suppresses lytic replication, E2F1 depletion markedly accelerates this process. Mechanistically, we establish that E2F1 and the lytic transactivator BZLF1 form a negative transcriptional feedback loop, tightly controlling viral lytic replication. Furthermore, E2F1 positively regulates c-Myc expression and together they repress the leaky BZLF1 expression during latency. Importantly, c-Myc does not influence E2F1 expression, nor does BZLF1 modulate c-Myc transcription, underlining a distinct regulatory hierarchy. In sum, our findings reveal that EBV tightly controls the latent-to-lytic switch through precise regulation of E2F1 expression, positioning E2F1 as a pivotal regulator of both cellular and viral gene expression. Synopsis EBV coordinates the latent-to-lytic switch by sensing E2F1 abundance, which acts as a crucial transcriptional regulator of both cellular and viral gene expressions. During EBV latent infection, E2F1 promotes c-Myc transcription, and together they suppress EBV lytic cycle transactivator BZLF1 expression. E2F1 and BZLF1 form a negative feedback loop in order to control each other’s transcriptions. BZLF1-driven controlled E2F1 expression successively inhibits c-Myc level, thereby stimulating EBV lytic cycle reactivation. BZLF1 does not regulate c-Myc, nor does c-Myc reciprocally regulate E2F1, emphasizing a unidirectional regulatory hierarchy.

Modulator agents that restore cystic fibrosis transmembrane conductance regulator (CFTR) function have revolutionized outcomes in cystic fibrosis, an incurable multisystem disease. Barriers exist to modulator use, making local CFTR gene and cell therapies attractive, especially in the respiratory tract. We used CRISPR to gene-correct CFTR in upper airway basal stem cells (UABCs) and show durable local engraftment into recipient murine respiratory epithelium. Interestingly, the human cells recapitulate the in vivo organization and differentiation of human sinus epithelium, with little expansion or contraction of the engrafted population over time, while retaining expression of the CFTR transgene. Our results indicate that human airway stem cell transplantation with locoregional restoration of CFTR function is a feasible approach for treating CF and potentially other diseases of the respiratory tract.

A key challenge in cancer research is to identify the secreted factors that contribute to tumor cell survival. Nowhere is this more evident than in Hodgkin lymphoma, where malignant Hodgkin Reed Sternberg (HRS) cells comprise only 1-5% of the tumor mass, the remainder being infiltrating immune cells that presumably are required for the survival of the HRS cells. Until now, there has been no way to characterize the complex Hodgkin lymphoma tumor microenvironment at genome scale. Here, we performed genome-wide transcriptional profiling with spatial and single-cell resolution. We show that the neighborhood surrounding HRS cells forms a distinct niche involving 31 immune and stromal cell types and is enriched in CD4+ T cells, myeloid and follicular dendritic cells, while being depleted of plasma cells. Moreover, we used machine learning to nominate ligand-receptor pairs enriched in the HRS cell niche. Specifically, we identified IL13 as a candidate survival factor. In support of this hypothesis, recombinant IL13 augmented the proliferation of HRS cells in vitro . In addition, genome-wide CRISPR/Cas9 loss-of-function studies across more than 1,000 human cancer cell lines showed that IL4R and IL13RA1, the heterodimeric partners that constitute the IL13 receptor, were uniquely required for the survival of HRS cells. Moreover, monoclonal antibodies targeting either IL4R or IL13R phenocopied the genetic loss of function studies. IL13-targeting antibodies are already FDA-approved for atopic dermatitis, suggesting that clinical trials testing such agents should be explored in patients with Hodgkin lymphoma.

Hedgehog (Hh) proteins elicit dose-dependent transcriptional responses by binding Patched receptors to activate transmembrane Smoothened (Smo) proteins. Activated Smo inhibits Ci/Gli transcription factor phosphorylation by Protein Kinase A (PKA) and consequent proteolytic processing to repressor forms; it also promotes nuclear transport and activity of full-length Ci/Gli proteins to induce Hh target genes. Smo-activated Fused (Fu) kinase drives Ci activation in Drosophila, while Suppressor of Fused (Su(fu)) counters full-length Ci/Gli activity and stabilizes full-length Ci/Gli by direct binding to at least three surfaces. Here, we used CRISPR-generated designer ci alleles to investigate alterations to Fu phosphorylation sites and to regions around Ci-Su(fu) interfaces under physiological conditions in Drosophila imaginal wing discs. Surprisingly, we identified alterations that activate Ci without significant loss of stabilization by Su(fu) and contributions of multiple Fu target sites to Ci activation in the absence of Su(fu), suggesting that the affected sites mediate Ci activation by regulating Ci-Ci, rather than Ci-Su(fu) interactions. We propose that those interactions maintain full-length Ci in a closed conformation that also facilitates, and is stabilized by, cooperative Ci-Su(fu) binding. Access to binding partners necessary for Ci activation is promoted through phosphorylation of at least four Fu sites on Ci, likely by directly disrupting Ci-Ci contacts and one Ci-Su(fu) interface without substantial Ci-Su(fu) dissociation, contrary to previous proposals. We also found that the Ci binding partner, Costal 2 (Cos2), which silences Ci in the absence of Hh, can facilitate Ci activation by Fu kinase.

Recent advances in single-cell DNA sequencing (scDNA-seq) and CRISPR technology have revolutionized gene therapy and drug discovery. However, data analysis requires expensive high-performance computing (HPC) clusters or large data servers, limiting reanalysis due to the lack of open-source software. To address this, we present scEDIT, a fast, lightweight, portable, and standalone software for pre- and post-processing CRISPR editing data from the Tapestri single-cell DNA-seq platform. scEDIT is memory-efficient, multithreaded, and compatible with most UNIX based systems. Tests using a low-cost desktop and public single cell CRISPR data demonstrate that the tool can efficiently process raw sequences, identify cell barcodes, count unedited and edited amplicons per cell, and outputs detailed filtered reads. Analysis of the single cell CRISPR data reveals indel patterns shared between in vitro experiments and unique indel profiles detected for in vivo study. Results further demonstrate the ability of single cell analysis in providing quantitative insights into the true zygosity of edited cell population. Although data shows a linear relation between indel frequencies by read count and cell count details of indel share between difference cells can only be truly explored with single cell data. The efficiency, stability, and portability of scEDIT makes it an invaluable tool for uncovering new insights into the single cell data without requiring expensive computational resources.

Conjugative plasmids are the main vehicle for the spread of antimicrobial resistance (AMR) genes in clinical bacteria. AMR plasmids allow bacteria to survive antibiotic treatments, but they also produce physiological alterations in their hosts that commonly translate into fitness costs. Despite the key role of plasmid-associated fitness effects in AMR evolution, their origin and molecular bases remain poorly understood. In this study, we introduce plasmid-wide CRISPR interference (CRISPRi) screens as a tool to dissect plasmid-associated fitness effects. We designed and performed CRISPRi screens targeting the globally distributed carbapenem resistance plasmid pOXA-48 in 13 different multidrug resistant clinical enterobacteria. Our results revealed that pOXA-48 gene-level effects are conserved across clinical strains, and exposed the key role of the carbapenemase-encoding gene, bla OXA-48 , as the main responsible for pOXA-48 fitness costs. Moreover, our results highlighted the relevance of postsegregational killing systems in pOXA-48 vertical transmission, and uncovered new genes implicated in pOXA-48 stability. This study sheds new light on the biology and evolution of carbapenem resistant enterobacteria and endorses CRISPRi screens as a powerful method for studying plasmid-mediated AMR.

The nuclear envelope (NE) is important for cellular health as it protects and organizes the genome. NE dynamics is important for various cellular processes including cell growth, migration and removal of defective NE components. In extreme cases, the NE can rupture leading to exchange of material between the nuclear interior and the cytoplasm. Rapid repair of the NE is initiated to minimize the effect on the genome. While our understanding of the machinery involved in this repair process is increasing, a lot is still unknown about this process including events leading up to NE rupture. Interestingly, biomolecular condensates have recently been found to play important roles in membrane repair and remodelling in cells. Here, we found that promyelocytic leukemia protein isoform II (PMLII), a protein involved in nuclear PML body formation, forms condensates at the NE. These condensates specifically form at sites where the lamina is disrupted. We show that NE rupture often occurs at these sites and that PMLII stays present until rupture repair is initiated suggesting a role in stabilization of the site for effective repair.

Mutations in the FA pathway lead to a rare genetic disease that increases risk of bone marrow failure, acute myeloid leukemia, and solid tumors. FA patients have a 500 to 800-fold increase in head and neck squamous cell carcinoma compared to the general population and the treatment for these malignancies are ineffective and limited due to the deficiency in DNA damage repair. Using unbiased CRISPR-interference screening, we found the loss of FA function renders cells dependent on key exocytosis genes such as SNAP23. Further investigation revealed that loss of FA pathway function induced deficiencies in lysosomal health, dysregulation of autophagy and increased lysosomal exocytosis. The compromised cellular state caused by the loss of FA genes is accompanied with decreased lysosome abundance and increased lysosomal membrane permeabilization in cells. We found these signatures in vitro across multiple cell types and cell lines and in clinically relevant FA patient cancers. Our findings are the first to connect the FA pathway to lysosomal exocytosis and thus expands our understanding of FA as a disease and of induced dependencies in FA mutant cancers.

ABSTRACT To identify new therapeutic targets that limit glioblastoma (GBM) invasion, we applied druggable-genome CRISPR screens to patient-derived GBM cells in micro-dissectible biomimetic 3D hydrogel platforms that permit separation and independent analysis of core vs. invasive fractions. We identified 12 targets whose suppression limited invasion, of which ACP1 (LMW-PTP) and Aurora Kinase B (AURKB) were validated in neurosphere assays. Proximity labeling analysis identified cortactin as an ACP1- AURKB link, as cortactin undergoes serine phosphorylation by AURKB and tyrosine dephosphorylation by ACP1. Suppression of ACP1 or AURKB in culture and in vivo shifted the balance of cortactin phosphorylation in GBM and reduced actin polymerization and actin-cortactin co-localization. Additional biophysical analysis implicated AURKB in GBM cell adhesion and cortical stiffness, and ACP1 in resistance to mechanical stress and shape plasticity needed for 3D migration. These findings reveal a novel targetable axis that balances kinase and phosphatase activities to regulate actin polymerization during GBM invasion.

Sorghum bicolor L. (Moench) ranks as the fifth most important crop globally, serving diverse purposes such as forage, food, feed, and fuel. Its natural tolerance to various environmental stresses makes it a valuable resource for addressing climate resilience. Harnessing such genetic resources for crop improvement requires highly efficient genetic transformation and genome editing tools. However, sorghum has historically been recalcitrant to genetic transformation, and publicly available engineering tools remain limited. In this study, we report a high-efficiency engineering toolkit, including shuttle and binary vectors, optimized for genome editing via Agrobacterium -mediated transformation. Using this toolkit, we successfully introduced CRISPR/Cas9-based editing machinery to target the phytoene desaturase ( PDS ) gene, achieving editing efficiencies up to 95.7% with monocistronic gRNA expression cassettes. To further expand the genome editing repertoire, we employed a protospacer adjacent motif (PAM)-flexible Cas9 variant, SpRY ( ZmSpRYi ), and achieved comparable editing efficiencies. Using our toolkit increases the potential for advanced editing tools to be used in engineering genetic variation of agronomically important quantitative traits. It also paves the way for deploying advanced genome editors, such as prime editors and base editors, to manipulate genetic and genomic resources. These advances provide a robust approach for advancing research in sorghum and other cereals, offering new opportunities for sustainable crop improvement.

We report the engineering of lipid nanoparticles (LNPs) to transport CRISPR/Cas9 payloads, including double-stranded DNA (dsDNA) donor templates, designed for homology directed repair (HDR)-mediated site-specific insertion of the cystic fibrosis transmembrane conductance regulator ( CFTR ) gene to correct cystic fibrosis (CF) in diseased airway epithelium. We screened various nanoparticle formulations, adjusting ratios of Cas9-encoding mRNA, single guide RNAs (sgRNAs), and dsDNA donor templates to optimize gene editing using human bronchial epithelial cells (16HBE14o-) harboring a CF-causing mutation (G542X). Populations of G542X cells edited via LNP delivery of CFTR donors achieved 3 – 3.5% gene integration and yielded comparable CFTR protein expression compared to normal 16HBE14o- controls. These edited populations exhibit restoration of CFTR-dependent Cl- current to ca. 80% of values measured in normal 16HBE14o- cell monolayers. This LNP platform adds capabilities for transporting large gene editing machinery to airway epithelial cells for genomic integration of entire genes, enabling therapeutic solutions that achieve correction of any CF-causing mutation.

ABSTRACT Allogeneic stem cell transplant (alloSCT) for patients with relapsed/refractory chronic lymphocytic leukemia (CLL) can result in cure in some patients. Analogous to chemotherapy and targeted therapy, we hypothesized that allogeneic cellular immunotherapies, including alloSCT and donor lymphocyte infusion (DLI), would impact malignant evolution through the application of selective immunologic pressure with reciprocal changes in the T cell compartment. We tested a cohort of 24 patients treated with HLA-matched alloSCT +/− DLI, two mediators of the graft versus leukemia (GVL) effect. Comparison of pre-alloSCT samples revealed that a key difference between responders (n=13) and non-responders (n=11) is the cellularity of leukemic cells. We further mapped mutational trajectories of tumor cells by whole exome sequencing (WES) of sort-purified CLL in 11 post-transplant relapsed patients and found evidence of subclonal leukemic evolution in 8/11 patients after nonmyeloablative human leukocyte antigen (HLA)-matched alloSCT. Different patterns of CLL evolution were observed, and these changes included putative CLL drivers in every case. To investigate the presence of immune-related variants in patients, we collected 19 T cell co-culture CRISPR datasets and identified the top positive and negative regulators of cancer cell’s response to T-cell-dependent killing. We found that most mutations linked to T-cell killing emerged after allo-SCT treatment, suggesting that selective pressures from the GVL effect may drive the evolution of these mutations. Together, these data identify cellular homogeneity as a key biomarker for susceptibility to GVL-driven immunity in CLL and illustrate how the leukemic cells further evolve to evade immunosurveillance. SIGNIFICANCE The impact of allogeneic stem cell transplant (alloSCT) on subclonal leukemia evolution remains poorly understood. By performing whole exome sequencing (WES) on pre- and post-alloSCT patient samples, we reveal different patterns of CLL evolution and potential immune-related driver genes influencing CLL relapse and refractory disease post-alloSCT.