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.

Protein knockdown using an improved auxin-inducible degron (AID2) technology has proven to be a powerful tool for studying protein function. The current approach requires the fusion of target proteins with a degron tag, a process typically achieved through CRISPR knock-in. However, knock-in remains challenging in non-model organisms and humans, limiting the broader applicability of AID2. To overcome this limitation, we developed a single-chain antibody AID2 (scAb-AID2) system. This approach employs an adaptor composed of a single-chain antibody fused with a degron, which recognises a target protein and induces rapid degradation in the presence of the inducer 5-Ph-IAA. We demonstrated that scAb-AID2, in combination with an anti-GFP nanobody, degraded GFP-fused proteins in human cells and C. elegans . Furthermore, we showed that endogenous p53 and H/K-RAS were conditionally degraded in cells expressing an adaptor encoding an anti-p53 nanobody and -RAS monobody, respectively, and led to aphidicolin sensitivity in cell culture and growth inhibition in mouse xenografts. This study paves the way for broader application of AID2-based target depletion in model and non-model organisms and for advancing therapeutic strategies.

Coding and enhancer variants of the RET receptor tyrosine kinase gene contribute to ∼50% of Hirschsprung disease (HSCR) risk, a congenital disorder of disrupted enteric nervous system (ENS) development. The greatest contribution of this risk is from a common variant (rs2435357) in an ENS-active, SOX10-bound RET enhancer (MCS+9.7) that reduces RET gene expression in vivo and triggers expression changes in other ENS genes in the human fetal gut. To uncover the cellular basis of RET -mediated aganglionosis, we used CRISPR/Cas9 to delete (Δ) the homologous mouse enhancer (mcs+9.7). We used single cell RNA sequencing and high-resolution immunofluorescence to demonstrate four significant features of the developing E14.5 gut of Δmcs+9.7/Δmcs+9.7 embryos: (1) a small (5%) yet significant reduction in Ret gene expression in only two major cell types – early differentiating neurons and fate-restricted inhibitory motor neurons; (2) no significant cellular loss in the ENS; and, (3) loss of expression of 19 cell cycle regulator genes suggesting a proliferative defect. To identify the Ret functional threshold for normal ENS development, we also generated, in combination with the Ret CFP null allele, (4) Δmcs+9.7/CFP double heterozygote mice which reduced Ret gene expression in the ENS to 42% with severe loss of inhibitory motor neurons, an effect restricted to the hindgut and driven by proliferative loss. Thus, Ret gene expression drives proliferation of ENS progenitor cells and hindgut-specific inhibitory motor neuron development, and that HSCR aganglionosis arises from a cascade of cellular defects triggered by >50% loss of Ret function.

Background Alpha-1 antitrypsin deficiency (A1ATD) is a hereditary recessive disorder caused by mutations in the SERPINA1 gene. It is a clinically under-recognised disease characterised by low circulating A1AT levels and intracellular accumulation of misfolded A1AT in hepatocytes. Deposition of excessive abnormal A1AT in the liver leads to liver failure, yet no specific treatments are available due to the lack of physiologically relevant disease modelling platforms. Methods We have hypothesised that human induced pluripotent stem cell (iPSC)-derived hepatocytes can provide an efficient platform to study A1ATD. Using CRISPR/Cas9, we have generated wild-type and A1ATD iPSC-derived hepatocytes (Opti-HEP) from healthy and A1ATD donors and developed a bioassay that mimics the accumulation of misfolded A1AT in the liver. Responses to the reference drug carbamazepine (CBZ), known to reduce intracellular misfolded A1AT levels, and RNA-based therapeutics were subsequently investigated. Results All lines successfully differentiated into hepatocytes as measured by comparable key hepatic and disease markers to those seen in primary human hepatocytes. The diseased lines displayed increased intracellular accumulation of misfolded A1AT compared to isogenic controls. Diseased cell lines showed significant decreases in intracellular accumulation of polymeric A1AT following transfection with RNA-based therapeutics, but a differential response upon treatment with CBZ. Conclusion We have developed a specific and robust in vitro model of A1ATD that recapitulates disease pathophysiology and responds to small molecule-based treatments and advanced therapeutic strategies. These data demonstrate the suitability of this model for large-scale efficacy screening studies for the treatment of A1ATD and help pave the way towards the development of novel therapies.

Plant volatiles shape plant-plant interactions by acting as defense regulators and response factors. While plant volatile biosynthesis is well understood, how their emission is regulated remains largely elusive. Here, we show that small peptide signaling regulates induced volatile release in maize. Following herbivore attack, green leaf volatiles such as ( Z )-3-hexenyl acetate (HAC) are released and induce terpene and indole emissions from neighboring plants. This process is accompanied by reduced expression of the ZmCLE1E9 gene and the ZmBAM1A, ZmBAM1B and ZmBAM3C receptor genes in HAC-exposed plants. Exogenous ZmCLE1E9 peptide inhibits HAC-triggered volatile release by limiting stomatal aperture. This inhibition disappears in the Zmbam1a/Zmbam1b/Zmbam3c triple mutant. Molecular docking supports ZmCLE1E9 and ZmBAMs as ligand-receptor pairs. Furthermore, Zmcle1e9 and Zmbams triple mutants show increased volatile emissions upon HAC exposure. In summary, we show that upon HAC perception, maize plants enhance their capacity to release terpenes and indole via the suppression of CLE1E9 signaling. This behavior allows maize plants to rapidly deploy volatile cues in response to stress volatiles and thus shape the infochemical dynamics of multitrophic environments.

ABSTRACT GABBR1 and GABBR2 are widely expressed in the brain and genetic inhibition of their function leads to widespread neurologic dysfunction and premature death in mice. Given that GABBR1 and GABBR2 heterodimerize to form a functional receptor, global knockout of GABBR1 or GABBR2 results in a similar phenotype, characterized by spontaneous epileptiform activity, hyperlocomotor activity, hyperalgesia, impaired memory and premature death. It is now known that both GABBR1 and GABBR2 are expressed in a variety of tissues outside the nervous system and that GABA-B receptors can heterodimerize with other class C GPCRs, including the extracellular calcium-sensing receptor (CaSR). Studies in vitro have demonstrated that interactions with GABBR1 and GABBR2 can alter CaSR signaling in human embryonic kidney cells and breast cancer cells. The neurologic consequences of global loss of function of GABBR1 or GABBR2 has made it difficult to study the effects of loss of GABBR function in other organs. While a conditional knockout for GABBR1 is available, the GABBR2 gene had not been “floxed”. We have used CRISPR to insert loxP sites into the GABBR2 locus in mice. These mice are normal at baseline but when bred with mice expressing Cre-recombinase under the control of the ubiquitously expressed Actin gene promoter, they recapitulate the phenotype of global GABBR2 knockout mice. Phenotypic changes through the brain, including the cortex, hippocampus and cerebellum. Evidence of abnormal neuronal function, increase cell death, and changes in neuronal architecture are seen throughout the brain of CRISPR knockout mice. These mice should be useful tools to study cell type-specific loss of GABBR2 function in the brain and other organs.

Spatial restriction of Aurora B to centromeres during prometaphase and metaphase enables it to phosphorylate proteins necessary for spindle assembly checkpoint signalling and biorientation of chromosomes on the mitotic spindle. Aurora B binding to T3-phosphorylated histone H3 (H3pT3) nucleosomes requires a multivalent targeting module, the chromosomal passenger complex (CPC), consisting of survivin, borealin, and INCENP. To shed light on how these components mediate CPC localisation during prometaphase and metaphase, we determined the structure of the CPC targeting module in complex with haspin-phosphorylated H3pT3-nucleosomes by cryo-electron microscopy. This structure shows how the N-terminus of borealin and the survivin BIR domain act as pivot and flexible tethering points, respectively, to increase CPC affinity for H3pT3 nucleosomes without limiting it to a specific orientation. We demonstrate that this flexible, yet constrained pivot-tether arrangement is important for the control of spindle assembly checkpoint signalling by Aurora B.

SUMMARY The surprising number and functional diversity of genes implicated in autism spectrum disorder (ASD) has made it challenging to identify core pathophysiological mechanisms or envision interventions with broad therapeutic potential. Here, parallel CRISPR-Cas13-based knockdown of 28 ASD genes and neighboring long non-coding RNAs reveals striking convergence on shared transcriptomic effects and neurodevelopmental phenotypes in human neural progenitor cells and cerebral organoids. Perturbations of single ASD genes caused the widespread dysregulation of other ASD genes, and de novo reconstruction of gene regulatory networks uncovered the prominent autism risk gene CHD8 as a critical driver of this transcriptomic convergence. The transcriptional activator ZFX , which escapes X chromosome inactivation in females, was also identified as a key regulator of ASD genes, revealing genetic underpinnings of the female protective effect. Thus, this study provides a crucial framework for uncovering how variants in diverse genes can cause convergent pathophysiological effects that ultimately result in a shared diagnosis.

Summary Autoimmunity develops as a result of a breakdown in immune tolerance and activation of autoreactive immune cells. Most of the common autoimmune diseases are polygenic 1 suggesting dysregulation in multiple signalling pathways. By contrast, in monogenic Inborn Errors of Immunity (IEI), which also can result in autoimmunity, the disease is triggered by a single genetic defect. Therefore, the discovery of causative mutations in IEI allows tracing the molecular mechanisms leading to autoimmunity in humans from a defect in the function of a specific gene to patients’ clinical and immunological phenotype. Here, we discovered an IEI patient with systemic autoimmunity caused by a private homozygous protein-truncating mutation in gene ZC3H12A leading to deficiency of Regnase-1, a regulatory RNase 2–5 . Flow cytometry, bulk T cell transcriptome analysis and single-cell RNA sequencing demonstrated expansion of γδ T cells expressing VCAM-1 and IFNγ genes. We show that Regnase-1 directly targets 3’UTR of VCAM1 and the coding sequence of IFNG mRNAs. These findings highlight a new autoimmunity mechanism in humans, where Regnase-1 deficiency causes expansion of VCAM1 + IFNG + T cells and their interaction with integrin α4β1-expressing B cells, which showed upregulation of IFN-response genes and activation, leading to systemic autoimmunity. Furthermore, we show that VCAM1+ T cells are present in organs of donors and are expanded in the blood of patients with systemic lupus erythematosus, a common autoimmune disease characterised by systemic autoimmunity.

ABSTRACT Primary human cells cultured in organoid format have great promise as potential regenerative cellular therapies. However, their immunogenicity and mutational profile remain unresolved, impeding effective long-term translation to the clinic. In this study we report, for the first time, the generation of human leukocyte antigen (HLA)-I and HLA-II knock-out expandable human primary cholangiocyte organoids (PCOs) using CRISPR-Cas9 as a potential ‘universal’ low-immunogenic therapy for bile duct disorders. HLA-edited PCOs (ePCOs) displayed the same phenotypical and functional characteristics as parental un-edited PCOs. Despite minimal off-target edits, single-molecule DNA-sequencing demonstrated that ePCOs and PCOs acquire substantial mutations in culture at similar rates but without evident selection for cancer-driver mutations. ePCOs induced reduced T cell-mediated immunity and a donor-dependent NK cell cytotoxicity in vitro and evaded cytotoxic responses with increased graft survival in humanized mice in vivo . Our findings have important implications for assessment of safety and immunogenicity of organoid cellular therapies.

Base-editing technologies, particularly cytosine base editors (CBEs), allow precise gene modification without introducing double-strand breaks; however, unintended RNA off-target effects remain a critical concern and are under-studied. To address this gap, we developed PiCTURE, a standardized computational pipeline for detecting and quantifying transcriptome-wide CBE-induced RNA off-target events. PiCTURE identifies both canonical ACW (W = A or T/U) motif-dependent and non-canonical RNA off-targets, revealing a broader WCW motif that underlies many unanticipated edits. Additionally, we developed two machine learning models based on the DNABERT-2 language model, termed STL and SNL, which outperformed motif-only approaches in terms of accuracy, precision, recall, and F1 score. To demonstrate the practical application of our predictive model for CBE-induced RNA off-target risk, we integrated PiCTURE outputs with the PROTECTiO pipeline and estimated RNA off-target risk for each transcript showing tissue-specific expression. The analysis revealed differences among tissues: while the brain and ovaries exhibited relatively low off-target burden, the colon and lungs displayed relatively high risks. Our study provides a comprehensive frame-work for RNA off-target profiling, emphasizing the importance of advanced machine learning-based classifiers in CBE safety evaluations and offering valuable insights for the development of safer genome-editing therapies.

ABSTRACT Patients with Acute Myeloid Leukemia (AML) subtypes, acute erythroleukemia and acute megakaryocytic leukemia (M6 and M7 AMLs, respectively) have a median survival of only a few months with no targeted effective treatment. Our gene expression analysis using the Cancer Cell Line Encyclopedia and CRISPR screen from DepMap showed that M6/M7 AMLs have high levels of the transcription factor GATA1 and depend on GATA1 for survival. While GATA1 was shown to support AML cell proliferation and resistance to chemotherapy, GATA1 has long been considered “undruggable”. Here, we identify the small molecule N-(4-hydroxyphenyl)retinamide (4-HPR, Fenretinide) as a novel GATA1 targeting agent in M6 and M7 AML cells, with nM to low μM concentrations of 4-HPR causing loss of GATA1. In M6 AML OCIM1 cells, knock-down of GATA1 induced cytotoxicity similarly to low doses 4-HPR while overexpression of GATA1 significantly protected cells from 4-HPR-induced cytotoxicity. In M6 AML cells resistant to current standard-of-care (SOC) Azacytidine plus Venetoclax, 4-HPR synergized with SOC overcoming cell resistance to the drugs. As single-agent, 4-HPR outperformed SOC. In M6 AML cells sensitive to SOC, 4-HPR enhanced and prolonged the growth inhibitory effect of SOC. 4-HPR is a synthetic derivative of vitamin A, and numerous clinical trials have supported its safe profile in cancer patients; therefore, targeted use of 4-HPR against M6 and M7 AMLs may represent a novel therapeutic window. Key Points - Fenretinide (4-HPR) targets the transcription factor GATA1, which was previously thought to be “undruggable” and induces GATA1 loss. - M6 and M7 Acute Myeloid Leukemias (AML) have enriched expression of GATA1 and they can be considered GATA1 positive. - Loss of GATA1 contributes significantly to 4-HPR cytotoxicity in M6 OCIM1 cells. - 4-HPR treatment overcomes chemotherapeutic resistance in M6 Acute Myeloid Leukemia cells, synergizes with standard-of-care and outperforms standard-of-care as a single agent.

ABSTRACT Fast twitch, type II muscle fibers are particularly prone to degradation in skeletal muscle pathologies, such as sarcopenia and muscular dystrophies. We previously showed that endogenous activation of the exercise-induced long noncoding RNA CYTOR promotes fast-twitch myogenesis. In the present study, we identify an independent pro-myogenic element within human CYTOR and optimize its RNA delivery. In human primary myoblasts exogenous, vector-based CYTOR exon 2 recapitulates the effect of full-length CYTOR by enhancing fast-twitch myogenic differentiation. Furthermore, chemically modified CYTOR exon 2 RNA ΨU (N1-me-PseudoU, 7-methyl guanosine 5’Cap, polyA tail) enhanced RNA stability and reduced the immunogenic response to CYTOR exon 2 RNA. We demonstrate that viral- or chemically optimized RNA-mediated CYTOR exon 2 administration enhances the commitment towards myogenic maturation in Duchenne muscular dystrophy-derived primary myoblasts, induced myogenic progenitor cells and mouse embryonic stem cells. Furthermore, chemically optimized CYTOR exon 2 improves key disease characteristics in dystrophic myotubes, including calcium handling and mitochondrial bioenergetics. In summary, our findings identify CYTOR exon 2 as the pro-myogenic domain of CYTOR that can be delivered in a disease context using chemical modifications. This is of particular importance given the susceptibility of type II muscle fibers in different muscle pathologies such as aging and dystrophies, and the reported oncogenic effect of CYTOR exon 1. Our study, therefore, highlights the potential of identifying functional domains in noncoding RNAs. Delivery, or targeting of such RNA domains could constitute next-generation RNA therapeutics.

The nucleoli are subdomains of the nucleus that form around actively transcribed ribosomal RNA (rRNA) genes. The highly repetitive and transcribed nature of the rRNA genes (rDNA) by RNA polymerase I (Pol I) poses a challenge for DNA repair and replication machineries. Here, we profile the nucleolar proteome and the chromatin landscape of stalled replication sites upon rDNA damage to characterize the early steps of nucleolar DNA damage response (nDDR). We observed early dynamics in nucleolar-nucleoplasmic proteome localization and identified nucleolar replication stress signatures involving chromatin remodeling networks, transcription-replication conflicts and DNA repair. Our findings define localized surveillance mechanisms that activate the nDDR. Further, we identified that upon rDNA damage, nucleolar RNA Polymerase (Pol) II binds to intergenic rDNA sequences and generates R-loops (DNA:RNA hybrid structures) that are essential for recruiting nDDR factors. Using a boutique CRISPR-Cas9 synthetic lethal screen of DNA repair factors with inhibitors of RNA Pol I transcription, we identified an unexpected protective role for the DNA translocase RAD54L in nDDR. Loss of Rad54L increases nucleolar R-loops and rDNA damage leading to defects in nucleolar structure and enhanced sensitivity to PARP and RNA Pol I inhibitors. Altogether, our study uncovers localized surveillance networks within the nucleolus that respond to rDNA damage. These insights expand our understanding of the molecular mechanisms governing nDDR and opens new avenues for developing nDDR-targeting therapies.

Plants employ diverse strategies to cope with different types of heat stress. The response to short-term acute heat stress differs significantly from that to moderate heat stress followed by severe stress events. After experiencing moderate heat stress, plants exhibit a more robust response to subsequent severe stress, a phenomenon known as thermopriming or acquired thermotolerance. Thermopriming creates a memory by maintaining the heat stress (HS) memory-related genes in an alert state. In this work, we investigated the role of Arabidopsis Universal Stress Protein 1 (USP1) in plant heat stress responses. CRISPR-Cas9 generated knockout usp1 mutant lines showed no morphological changes during development and normal growth conditions. However, usp1 mutant plants showed enhanced levels of apoplast hydrogen peroxide and superoxide reactive oxygen species accumulation upon heat stress. Transcriptome analyses revealed that genes related to protein folding, electron transport, and oxidative phosphorylation are strongly upregulated in usp1 mutant plants. USP1 is essential for acquired thermotolerance, as usp1 mutants are compromised in heat stress memory but show normal responses to acute heat stress similar to hsfa2 mutants. Biochemical assays showed that USP1 functions as a molecular chaperone, protecting the transcription factor HSFA2 from heat-induced denaturation. Moreover, usp1 mutant plants show decreased transcript levels of heat stress response genes and reduced H3K4me3 enrichment at memory gene loci. These data show that USP1 plays an important role as a chaperone of HSFA2 in mediating plant heat stress memory.