Single-guide RNA lentiviral infection with Cas9 protein electroporation (SLICE) enables CRISPR screening in primary cell types that require transient Cas9 expression yet is limited by scalability and robustness. Here, we introduce dual-guide RNA infection with Cas9 electroporation (DICE), which expresses two guides from the same lentiviral construct that target the same gene. In genome-wide screens, DICE outperformed SLICE in defining essential genes and modulators of PD-L1 expression in IFN gamma activated THP1 cells. Collectively, these data demonstrate that DICE can be utilized for reduced-scale CRISPR screens in cell types with transient Cas9 protein expression without sacrificing screening quality.

ABSTRACT Bacterial transcription initiation is a tightly regulated process that canonically relies on sequence-specific promoter recognition by dedicated sigma (σ) factors, leading to functional DNA engagement by RNA polymerase (RNAP) 1 . Although the seven σ factors in E. coli have been extensively characterized 2 , Bacteroidetes species encode dozens of specialized, extracytoplasmic function σ factors (σ E ) whose precise roles are unknown, pointing to additional layers of regulatory potential 3 . Here we uncover an unprecedented mechanism of RNA-guided gene activation involving the coordinated action of σ E factor in complex with nuclease-dead Cas12f (dCas12f). We screened a large set of genetically-linked dCas12f and σ E homologs in E. coli using RIP-seq and ChIP-seq experiments, revealing systems that exhibited robust guide RNA enrichment and DNA target binding with a minimal 5ʹ-G target-adjacent motif (TAM). Recruitment of σ E was dependent on dCas12f and guide RNA (gRNA), suggesting direct protein-protein interactions, and co-expression experiments demonstrated that the dCas12f-gRNA-σ E ternary complex was competent for programmable recruitment of the RNAP holoenzyme. Remarkably, dCas12f-RNA-σ E complexes drove potent gene expression in the absence of any requisite promoter motifs, with de novo transcription start sites defined exclusively by the relative distance from the dCas12f-mediated R-loop. Our findings highlight a new paradigm of RNA-guided transcription (RGT) that embodies natural features reminiscent of CRISPRa technology developed by humans 4,5 .

ABSTRACT RNA-guided proteins have emerged as critical transcriptional regulators in both natural and engineered biological systems by modulating RNA polymerase (RNAP) and its associated factors 1-5 . In bacteria, diverse clades of repurposed TnpB and CRISPR-associated proteins repress gene expression by blocking transcription initiation or elongation, enabling non-canonical modes of regulatory control and adaptive immunity 1,6,7 . Intriguingly, a distinct class of nuclease-dead Cas12f homologs (dCas12f) instead activates gene expression through its association with unique extracytoplasmic function sigma factors (σ E ) 8 , though the molecular basis has remained elusive. Here we reveal a novel mode of RNA-guided transcription initiation by determining cryo-electron microscopy structures of the dCas12f-σ E system from Flagellimonas taeanensis . We captured multiple conformational and compositional states, including the DNA-bound dCas12f-σ E -RNAP holoenzyme complex, revealing how RNA-guided DNA binding leads to σ E -RNAP recruitment and nascent mRNA synthesis at a precisely defined distance downstream of the R-loop. Rather than following the classical paradigm of σ E -dependent promoter recognition, these studies show that recognition of the −35 element is largely supplanted by CRISPR-Cas targeting, while the melted −10 element is stabilized through unusual stacking interactions rather than insertion into the typical recognition pocket. Collectively, this work provides high-resolution insights into an unexpected mechanism of RNA-guided transcription, expanding our understanding of bacterial gene regulation and opening new avenues for programmable transcriptional control.

ABSTRACT Oxytocin receptors (OTR) within the extended amygdala and nucleus accumbens have been implicated in modulating social behaviors, particularly following stress. The effects of OTR could be mediated by modulating the activity of pre-synaptic axon terminals or via post-synaptic neurons or glia. Using a viral-mediated CRISPR/Cas9 gene editing system in California mice ( Peromyscus californicus ), we selectively knocked down OTR in the anteromedial bed nucleus of the stria terminalis (BNST) or the nucleus accumbens (NAc) to examine their roles modulating social approach and vigilance behaviors. Knockdown of OTR in the BNST attenuated stress-induced decreases of social approach and increases of social vigilance behaviors in adult female California mice, similar to prior pharmacological studies. These effects were more prominent in the large arena social interaction where mice could control proximity to a social target with a barrier (wire cage). In a small arena interaction test where focal mice freely interacted with target mice, effects of BNST OTR knockdown were muted. This suggests that within the BNST OTR are more important for modulating behavioral responses to more distal stimuli versus more proximal social contexts. In mice with OTR knockdown in the NAc, few behavioral changes were observed which is consistent with previous findings of the importance of presynaptic OTR, which were unaffected by our gene editing strategy, driving social approach behaviors in the NAc. Interestingly, BNST OTR knockdown increased exploratory behavior toward a non-social stimulus after stress, pointing to a potentially broader role for BNST OTR function. Our findings highlight the region- and context-specific functions of OTR in social behavior and the advantages of using a selective gene-editing tool to dissect the neural circuits that influence social and stress-related responses.

Abstract The fall armyworm (FAW), Spodoptera frugiperda , is an invasive and polyphagous pest that has become a major threat to tropical crops due to its rapid dissemination, broad host range, and increasing insecticide resistance. This research approaches the molecular basis of resistance by combining comparative transcriptomics with RNA interference (RNAi) to discover and functionally validate critical detoxification genes. We identified significant up-regulation of cytochrome P450 monooxygenases (CYP321A1 and CYP9A4), glutathione S-transferases (GSTE6 and GSTE2) and carboxylesterase (CCE2) in resistant populations. Suppression of these genes through RNAi resulted in greater susceptibility to insecticides and lower larval survival. Delivery of the chitosan nanoparticle using dsRNA also improved stability and knockdown in a field context. Phylogenetic analysis revealed that these resistance genes are well conserved among the most important lepidopteran pests, indicating potential for a broader efficacy of RNAi as a pest management strategy. It may establish RNAi- and CRISPR-based molecular biocontrol systems in tropical IPMs, with significant benefits for sustainable agriculture and biodiversity conservation.

Gene editing technologies such as CRISPR/Cas9 have revolutionized functional genomics, yet their application in marine fish cell lines remains limited by inefficient delivery. This study compares three delivery strategies—electroporation, lipid nanoparticles (LNPs), and magnetofection using gelatin-coated superparamagnetic iron oxide nanoparticles (SPIONs)—for CRISPR/Cas9-mediated editing of the ifi27l2a gene in DLB-1 and SaB-1 cell lines. We evaluated transfection and editing efficiency, intracellular Cas9 localization, and genomic stability of the target locus. Electroporation achieved up to 95% editing in SaB-1 under optimized conditions, but only 30% in DLB-1, which exhibited locus-specific ge-nomic rearrangements. Diversa LNPs enabled intracellular delivery and moderate edi-ting (~25%) in DLB-1, while SPION-based magnetofection resulted in efficient uptake but no detectable editing, highlighting post-entry barriers. Confocal imaging and fluore-scence correlation spectroscopy revealed that nuclear localization and Cas9 aggregation are critical determinants of editing success. Our findings demonstrate that CRISPR/Cas9 delivery efficiency is cell line-dependent and governed by intracellular trafficking and genomic integrity. These insights provide a practical framework for optimizing gene editing in marine teleosts, advancing both basic research and selective breeding in aquaculture.

SUMMARY Neural crest induction begins early during neural plate formation, requiring precise transcriptional control to activate lineage-specific enhancers. Here, we demonstrate that SALL4, a transcription factor strongly expressed in cranial neural crest cells (CNCCs) and associated with syndromes featuring craniofacial anomalies, plays a critical role in this early regulatory process. Using a SALL4 -haploinsufficient human iPSC model that recapitulates clinical haploinsufficiency, we show that the SALL4A isoform directly interacts with the BAF complex subunit DPF2 through its zinc-finger-3 domain. This interaction enables SALL4-mediated recruitment of BAF to CNCC-specific enhancers during early neural plate stages. Without functional SALL4, BAF fails to load at chromatin, leaving CNCC enhancers inaccessible despite normal neuroectodermal progression. Consequently, cells cannot undergo proper CNCC induction and specification due to persistent enhancer repression. Our findings reveal SALL4 as an essential regulator of BAF-dependent enhancer activation during early neural crest development, providing molecular insights into SALL4-associated craniofacial anomalies.

Abstract Genome editing using CRISPR/Cas9 allows precise modifications within plants genomes, however, it also poses biosafety and biosecurity concerns, particularly regarding potential off-target mutations. Therefore, establishing robust detection methods is essential. A loop-mediated isothermal amplification (LAMP) assay coupled with Thioflavin T (ThT) fluorescence detection was developed for the sensitive and specific identification of the Cas9 coding sequence in genome-edited plants. Following optimization of reaction conditions—including ThT concentration, Mg²⁺ levels, and incubation time—the assay achieved a detection limit of approximately 4.34 copies/µL based on fluorescence analysis. Two G-rich sequences predicted from the LAMP amplicons were evaluated individually, revealing that one produced significantly higher fluorescence, suggesting a potential G-quadruplex (G4)-like conformation enhancing ThT signal. The method avoids colorimetric subjectivity and does not require real-time monitoring or complex instrumentation. Amplification products were confirmed via agarose gel electrophoresis, and fluorescence measurements were consistent across replicates. This study provided a isothermal assay to utilize ThT for Cas9 detection and offers a cost-effective, reliable platform for screening genome-edited organisms, particularly in settings with limited resources. The system shows strong potential for future application in biosafety and traceability.

Abstract CRISPR-Cas nucleases are transforming genome editing, RNA editing, and diagnostics but have been limited to RNA-guided systems. We present ΨDNA, a DNA-based guide for Cas12 enzymes, engineered for specific and efficient RNA targeting. ΨDNA mimics a crRNA but with a reverse orientation, enabling stable Cas12-RNA assembly and activating trans-cleavage without RNA components. ΨDNAs are effective in sensing short and long RNAs and demonstrated 100% accuracy for detecting HCV RNA in clinical samples. We discovered that ΨDNAs can guide certain Cas12 enzymes for RNA targeting in cells, enhancing mRNA degradation via ribosome stalling and enabling multiplex knockdown of multiple RNA transcripts. This study establishes ΨDNA as a robust alternative to RNA guides, augmenting CRISPR-Cas12’s potential for diagnostic applications and for targeted RNA modulation in cellular environments.

Abstract CRISPR/Cas effectors rely on RNA guides to recognize target nucleic acids. In Cas9 and Cas12a systems, protospacer adjacent motif (PAM) on target DNA engage conserved residues in the Cas protein, thereby accelerating target binding and catalytic activation. Here, we overturn this paradigm by reprograming the canonical RNA-guided DNA-targeting machinery into a DNA-guided RNA-targeting platform. We engineered synthetic CRISPR DNA (crDNA) that emulates the PAM-duplex architecture to form stable deoxyribonucleoprotein complexes with Cas9 or Cas12a, redirecting their specificity towards RNA substrates. Coupling DNA-guided Cas12a with isothermal amplification enables attomolar detection of nucleic acid samples, while DNA-guided Cas9 achieves over 70% knock-down of EGFP expression in HEK293T cells. This DNA-guided paradigm reshapes our understanding of CRISPR targeting and paves the way for new diagnostic and therapeutic applications.

Vascular malformations are congenital lesions caused by somatic and germline mutations that disrupt developmental signaling pathways. Capillary malformations (CMs) typically present as port-wine stains in the skin and can also affect ocular and cerebral tissues in Sturge Weber Syndrome (SWS), leading to aesthetic, ophthalmic, and neurological complications. CMs are caused by a somatic mutation in the GNAQ gene in endothelial cells, leading to a p.R183Q substitution in the Gαq protein. The underlying mechanisms of Gαq-R183Q-driven CMs formation remain unclear. To address this, we generated CRISPR/Cas9-engineered human dermal microvascular endothelial cells lacking endogenous Gαq, whilst expressing the Gαq-R183Q mutant instead. The Gαq-R183Q mutation strongly impaired endothelial cell migration and angiogenic sprouting capacity compared to wild-type controls. Next, using SILAC-based quantitative proteomics, we investigated the Gαq-R183Q-induced changes in the endothelial phosphoproteome. These analyses revealed prominent activation of the calcineurin-NFAT signaling pathway in Gαq-R183Q-expressing endothelial cells, leading to dephosphorylation of NFAT1 and NFAT2 and the selective expression of their transcriptional target DSCR1.4. Immunofluorescence of patient-derived skin biopsies confirmed deregulation of NFAT1/2 and the expression of DSRC1 in endothelial cells, validating their potential importance in CMs. We further demonstrate that pharmacological inhibition of calcineurin with tacrolimus (FK506) could partially restore NFAT signaling in Gαq-R183Q endothelial cells. Intriguingly, the genetic depletion of the NFAT target DSCR1 in Gαq-R183Q cells fully restored calcineurin/NFAT signaling to normal levels, enabling proper endothelial migration and sprouting. In summary, we uncovered a calcineurin-NFAT-DSCR1.4 signal transduction axis that is driven by Gαq-R183Q and established its importance for endothelial angiogenic properties. These findings highlight the calcineurin/NFAT signaling axis as a promising therapeutic target to restore endothelial function in CMs.

Flaviviruses such as dengue and Zika viruses extensively remodel host cell membranes to create specialised replication organelles, but the molecular mechanisms governing lipid metabolism during infection remain poorly understood. Through systematic screens of fatty acyl transferase enzymes (MBOAT and zDHHC families) and complementary approaches including CRISPR/Cas9 gene deletions, pharmacological inhibition, proteomics, and photo-crosslinkable cholesterol analogues, we identified Sterol O-acyltransferases 1 and 2 (SOAT1/SOAT2) as critical host dependency factors for flavivirus infection. SOAT1/2 activities were upregulated early during infection, coinciding with increased LD formation, which underwent transition to liquid crystalline phases. Genetic deletion or pharmacological inhibition of either enzyme resulted in a dramatic ∼100-fold reduction in viral production. Mechanistically, SOAT1/2 generate cholesteryl ester-enriched lipid droplets with fundamentally altered proteomes, enriched in fatty acid remodelling enzymes, Rab-GTPases, lipid transport proteins and sphingomyelinases. Photo-crosslinking experiments demonstrated direct interactions between LD-derived cholesterol and viral prM, capsid and NS1. SOAT1/2 deficiency resulted in defective, viral RNA-free replication organelles and complete absence of immature virions. Supporting the clinical relevance of viral lipid exploitation, analysis of dengue patients from a Sri Lankan cohort revealed that central obesity significantly increased the risk of severe dengue haemorrhagic fever, compared to lean patients. This study establishes SOAT1/2 as essential host factors that enable flavivirus morphogenesis through specialised cholesteryl ester-enriched LDs, revealing a previously unrecognised virus-host interaction mechanism and identifying host lipid metabolism as a promising therapeutic target for combating flavivirus infections.

ABSTRACT Nephronophthisis (NPH) is a heterogeneous, autosomal recessive ciliopathy and an important cause of end-stage renal disease (ESRD) in children and young adults. Since its classification as ciliopathy in 2003, NPH disease causal attribution had been focused primarily on ciliary dysfunction. The finding that ciliopathy players are involved in the DNA damage response (DDR) signaling resulted in a paradigm shift in thinking on NPH disease aetiology. Mutations in NPHP1 are the leading cause of NPH, but the underlying mechanisms that lead to the disease phenotype remain poorly understood. Here, nephrocystin-1 depleted kidney organoids were generated and characterized to address this knowledge gap. We used CRISPR/Cas9 to generate NPHP1 control ( NPHP1 WT ) and two mutant ( NPHP1 ko1 and NPHP1 ko2. ) cell lines from healthy human induced pluripotent stem cells (iPSC), differentiated into kidney organoids in an air-liquid interface following an optimized protocol. Upon loss of nephrocystin-1, kidney organoids showed impaired nephron structures and loss of glomerular mesangial and distal tubular cells. Furthermore, NPHP1 depleted organoids exhibited a persistent inability to repair DNA lesions and showed increased senescence and fibrosis characteristics. Dynamic subcellular localization of nephrocystin-1 in NPHP1 WT , particularly its translocation to nuclei 15 min post-UVC light exposure, suggested its direct involvement in the DDR. In conclusion, a novel NPHP1 -depleted kidney organoid model was established, providing a platform to comprehensively study DNA damage, senescence and fibrosis simultaneously upon nephrocystin-1 loss. This advanced model aids in the understanding of the pathophysiology of NPH and paves the way towards identifying novel druggable targets.

ABSTRACT Botrytis cinerea is recognized as one of the most harmful fungal diseases affecting grapevine ( Vitis vinifera ), directly impacting grape yield and wine quality. Identifying new genes involved in the interaction between V. vinifera and B. cinerea appears to be the most promising strategy for enhancing grapevine resistance to this pathogen in future breeding programs. During pathogen infection, plasma membrane-localized pattern recognition receptors (PRRs) are involved in the perception of conserved microbe-associated molecular patterns (MAMPs). Among PRRs, members of the LysM receptor-like kinase family are well known to mediate recognition of fungal MAMPs to induce the plant immune signaling pathway. Interestingly, a novel member of this receptor family, named VvLYK6, was identified in grapevine as the most upregulated during a Botrytis cinerea infection. Aim: ing to understand the role of VvLYK6 in plant immunity, we carried out an overexpression study in Arabidopsis thaliana and in grapevine cell suspension. The overexpression of VvLYK6 resulted in a reduction of chitin oligomer-induced MAPKs activation, expression of defense-related genes, a reduced callose deposition and an increased plant susceptibility to three fungal pathogens. At the opposite, the CRISPR-Cas9-mediated vvlyk6 knock-out lines generated in V. vinifera induce the phytoalexin-related stilbene pathway at the basal level. Based on our findings, we concluded that VvLYK6 negatively regulates the chitin-triggered immune responses in V. vinifera , suggesting its potential involvement as a susceptibility gene during fungal infections.

Abstract Molnupiravir, a nucleoside analog effective against RNA viruses such as Severe Acute Respiratory Syndrome Coronavirus type 2 (SARS-CoV-2), exerts antiviral effects primarily through lethal mutagenesis by incorporation into viral RNA. However, its ambiguous base-pairing properties raise concerns about potential mutagenesis via conversion to deoxycytidine diphosphate (dCDP) by cellular ribonucleotide reductase (RNR) and subsequent incorporation into DNA. This study evaluates whether herpes simplex virus type 1 (HSV-1), in comparison to HSV-1 lacking RNR (HSV-1 ΔRR), can serve as a model to investigate molnupiravir’s mutagenic effects on DNA viruses. Using CRISPR-Cas9, the ICP6 RNRdomain has already been deleted to generate HSV-1 ΔRR. Human primary fibroblasts with low endogenous deoxyribonucleotide pools were infected at low multiplicity and treated with varying concentrations of molnupiravir. Viral titers were measured by TCID50 assays, and viral DNA from treated cultures was sequenced, targeting nonessential genes to detect mutations. Molnupiravir significantly reduced viral titers of wild-type HSV-1 in both fibroblasts and Vero cells, while HSV-1 ΔRR exhibited reduced sensitivity, with significant inhibition only at higher drug concentrations in fibroblasts. Importantly, no mutations were detected in viral DNA from either strain at any molnupiravir concentration. These findings indicate that molnupiravir’s mutagenic potential is limited in DNA viruses like HSV-1, likely due to restricted incorporation into DNA. This highlights the need for further research to optimize molnupiravir’s antiviral use beyond RNA viruses.

Rationale Chimeric antigen receptor (CAR) T cell therapies have shown remarkable success in treating hematological cancers and are increasingly demonstrating potential for solid tumors. CRISPR-based genome editing offers a promising approach to enhance the potency and safety of CAR-T cells. However, several challenges persist, including inefficient tumor homing and treatment-related toxicities in normal tissues, which continue to hinder widespread adoption. Advanced imaging technologies, including bioluminescence imaging (BLI) and positron emission tomography (PET), provide real-time insights into CAR-T cell distribution and activity in vivo, both in preclinical models and in patients. Here, we developed Trackable Reporter Adaptable CRISPR-Edited CAR (tRACE-CAR) T cells, a modular system for site-specific integration of CARs and imaging reporters. Methods The luciferase reporter AkaLuciferase (AkaLuc) or the human sodium iodide symporter (NIS) were cloned downstream of the CAR in adeno-associated virus (AAV) donors for BLI or PET tracking, respectively. CARs with imaging reporters were knocked into the TRAC locus of primary human T cells via CRISPR editing and AAV transduction. Editing efficiency was evaluated by flow cytometry and junction PCR. In vitro cytotoxicity was assessed by BLI using firefly luciferase (Fluc)-expressing cancer cells co-cultured with CAR-T cells at varying effector-to-target ratios. In vivo, BLI and PET imaging assessed CAR-AkaLuc and CAR-NIS T cell expansion and trafficking in Nod-SCID-gamma mice bearing xenograft tumors. Results T cell receptor (TCR) knockout efficiency exceeded 85%, with CAR expression observed in 70–80% of cells, depending on the reporter used. Reporter-engineered CAR-T cells retained functionality in vitro and exhibited significant cytotoxicity against target cancer cells, outperforming naïve T cells. In vivo, AkaLuc BLI and 18 F-tetrafluoroborate PET enabled non-invasive tracking of viable CAR-T cells. Notably, the route of administration (intravenous, peritumoral, or intraperitoneal) significantly influenced the distribution of CAR-T cells and their therapeutic effectiveness. Conclusion tRACE-CAR enabled precise optical and PET tracking of CAR-T cells in models of B cell leukemia and ovarian cancer, allowing dynamic, non-invasive monitoring of cell distribution in both tumors and off-target tissues. This imaging platform could lead to more personalized, effective CRISPR-edited CAR cell therapies.

Functional regeneration of volumetric muscle loss (VML) remains a significant challenge in the field of regenerative medicine. We have developed a novel biomimetic scaffold (0.4 mm thick hernia patch) derived from soluble collagen. This biomimetic scaffold exhibits exceptional strength, low immunogenicity, and structurally mimics the natural muscle fiber arrangement. The scaffold is utilized to repair a VML rabbit model (30 × 30 mm defect in abdominal wall) using nylon sutures for connection. It was observed that the VML site was gradually covered by regenerated tissue, which consisted of 2-3 mm thick functional muscle. By 32 weeks post-surgery, the newly formed muscle tissue had covered the majority of the VML area, as evidenced by morphological observations and histological evaluations. Approximately 24 weeks after surgery, the scaffold underwent complete degradation. This degradation exhibited a strong correlation with muscle regeneration. Furthermore, it was observed that the nylon sutures gradually migrated towards the VML center as muscle regeneration progressed, and nylon sutures exhibited an adverse impact on muscle regeneration.. The study did not employ exogenous cells or growth factors. However, the collagen scaffold effectively stimulated endogenous muscle regeneration. In contrast to the previous limited partial recovery or fibrotic scar healing, the collagen scaffold successfully induced genuine structural and functional muscle regeneration. The 0.4 mm thick scaffold (hernia patch) exhibits a different structure from the 2-3 mm thick natural abdominal muscle wall. This suggests that a simple scaffold can regenerate functional tissue with a complex structure. This research represents the first successful functional regeneration of VML in clinically relevant animal models through the utilization of artificial materials. This technology possesses transformative potential in the treatment of muscle defects arising from trauma, tumor resection, hernia, or congenital anomalies. Furthermore, it holds substantial medical potentials in addressing neuromuscular diseases, such as Duchenne muscular dystrophy (DMD), amyotrophic lateral sclerosis (ALS), and spinal muscular atrophy (SMA), by collaborating with advanced technologies including CRISPR-Cas9, adeno-associated virus (AAV) gene therapy, and stem cell technology.

The compositions of conserved gene families often vary widely between species, complicating predictions and experimental tests of shared versus distinct functions, especially in families shaped by extensive duplication, redundancy, and paralog diversification. The plant CLV3/EMBRYO- SURROUNDING REGION ( CLE ) small-signaling peptide family exemplifies these challenges. Although genetic studies in model systems have identified shared roles for a few CLE genes and species-specific redundancies, an evolutionary analysis of the entire family over deep time could empower predictive and experimental dissections of functions obscured by redundancy. We developed a scanning pipeline that de novo annotated CLE genes from 2,000 genomes representing 1,000 species, uncovering thousands of previously undetected family members and producing a comprehensive phylogenetic reconstruction and tracing of the family’s evolution and sequence diversification over 140 million years. Computational modeling of coding and cis-regulatory regions predicted lineage-specific asymmetries in paralog redundancy, stemming from ancestral amino acids in the functional core of the dodecapeptide and partial conservation of promoter elements. We tested these predictions using two genome-editing strategies in Solanaceae. Base- editing of deeply conserved residues in the CLV3 dodecapeptide and its paralogs across three species confirmed their critical roles in repressing stem-cell proliferation, and multiplex CRISPR knockouts of the 52 tomato CLE genes resolved pairwise and higher-order redundancies, revealing previously uncharacterized regulators of shoot architecture and plant size. These findings show how both peptide and cis-regulatory erosion shape CLE redundancy and provide a framework for detecting and translating deep evolutionary signals into testable genetic hypotheses across compositionally complex gene families.

In Saccharomyces cerevisiae , glucose depletion induces metabolic reprogramming through widespread transcriptional and translational reorganization. We report that initial, very rapid translational silencing is driven by a specialized metabolic mechanism. Following glucose withdrawal, intracellular NTP levels drop drastically over 30 sec, before stabilizing at a regulated, post-stress set-point. Programmed translational control results from the differential NTP affinities of key enzymes; ATP falls below the (high) binding constants for DEAD-box helicase initiation factors, including eIF4A, driving mRNA release and blocking 80S assembly. Contrastingly, GTP levels always greatly exceed the (low) binding constants for elongation factors, allowing ribosome run-off and orderly translation shutdown. Translation initiation is immediately lost on all pre-existing mRNAs, before being preferentially re-established on newly synthesized, upregulated stress-response transcripts. We conclude that enzymatic constants are tuned for metabolic remodeling. This response counters energy depletion, rather than being glucose-specific, allowing hierarchical inhibition of energy-consuming processes on very rapid timescales.

ABSTRACT Pancreatic ductal adenocarcinoma (PDA) is a deadly malignancy with limited effective therapies. Adoptive cell therapy (ACT) is a promising treatment modality for patients with solid tumors but has been limited by the highly fibroinflammatory and immunosuppressive tumor microenvironment (TME). Transforming growth factor-β (TGFβ) participates in the inordinately suppressive TME in PDA. Here, we test the impact of selective Tgfbr2 deletion using CRISPR/Cas9 or genetic approaches in mesothelin (Msln)-specific T cell receptor (TCR) engineered T cells during ACT of PDA. Abrogating TGFβ signaling augmented TCR-engineered T cell accumulation in autochthonous and orthotopic PDA models and promoted terminal effector T cells, although this largely required inclusion of a vaccine at the time of T cell transfer. While loss of Tgfbr2 impaired CD103 upregulation, it only modestly impaired donor T cell central, tissue resident, or Tcf1 + Slamf6 + stem-like memory T cell formation. These attributes ultimately result in heightened functional capacity and delayed tumor growth. Unexpectedly, however, most tumor-infiltrating engineered T cells failed to differentiate into PD-1 + Lag3 + exhausted T cells (T EX ) regardless of TGFβR2 expression and despite abundant Msln protein expression by PDA cells. Forcing Msln epitope processing in KPC tumor cells promoted donor T cell accumulation, acquisition of PD-1 and Lag3, increased IFNγ production by TCR-engineered T cells refractory to TGFβ and bypassed the vaccine requirement for therapeutic efficacy. Thus, promoting increased antigen processing/presentation by tumor cells while abrogating Tgfbr2 in engineered T cells can sustain donor T cell function in the suppressive TME and enhance the therapeutic efficacy of ACT. Our study supports pursuit of strategies that modulate tumor intrinsic antigen processing while relieving T cell suppression to safely promote the antitumor activity of TCR-engineered T cells.

Elucidating the gene regulatory networks (GRNs) that govern human B cell differentiation is essential for understanding immune responses to infection, vaccination and autoantigens. Here, we show that individual naive B cells can give rise to both plasma cells and germinal centre (GC) B cells. In contrast, memory B cells display a progressive increase in IRF4 activity over time, leading to PRDM1 induction and exclusive differentiation into plasma cells. Using CRISPR-based perturbations, we demonstrate that IRF4 is indispensable for both GC and plasma cell development. Notably, IRF4 promotes GC fate independently of PRDM1, as PRDM1 disruption did not impair GC differentiation. We also find that while the abundance of antibody mRNAs is clonally correlated, class switch recombination (CSR) occurs stochastically and is clonally independent. Together, these findings reveal distinct regulatory dynamics during naive and memory B cell activation and offer new insights into the GRNs underlying human B cell fate decisions.

ABSTRACT Interindividual genetic variation associated with cardiovascular traits and disease is commonly found in the non-coding genome, suggesting regulatory changes underlie many genetic association signals. Mammalian gene regulation is controlled by promoters and enhancers with rapidly divergent activities. However, the interplay between human non- coding genetic variation and regulatory evolution remains largely unexplored. Here, we investigated promoter and enhancer evolution in the mammalian heart using genome-wide epigenomic profiling, intersected these elements with genetic variants associated with cardiovascular traits, and experimentally tested candidate regions in human cardiomyocytes derived from pluripotent stem cells. First, we applied a comparative genomics approach to identify epigenomically-conserved promoters and enhancers in the heart, as well as elements with primate-specific or human-only epigenomic signals. Second, we evaluated the association of common genetic variation and signatures of cell-type specific gene regulation with mammalian epigenomic conservation. We report an enrichment of common genetic variants in epigenomically-conserved promoters and enhancers, which also associate with regulatory pleiotropy across cardiac cell types, and promoter-enhancer contacts in cardiomyocytes. Based on these findings, we selected candidate epigenomically-conserved elements across three cardiovascular genetics loci ( KCNH2 , CPEB4 and PRKCE ), and investigated gene expression and cellular outcomes upon CRISPR/Cas9-mediated deletion of each region in human cardiomyocytes. These analyses inform how mammalian epigenomic conservation associates with specific gene expression contributions and downstream cellular responses, such as cardiomyocyte hypertrophy and sensitivity to hypoxia/reoxygenation. Moreover, the comparative analyses we present here contribute to ongoing efforts to prioritise and functionally characterise cardiovascular association signals from human population genetics.

ABSTRACT Aberrant biomolecular condensates are implicated in multiple incurable neurological disorders, including Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Dementia (FTD), and DYT1 dystonia. However, the role of condensates in driving disease etiology remains poorly understood. Here, we identify myeloid leukemia factor 2 (MLF2) as a disease-agnostic biomarker for phase transitions, including stress granules and nuclear condensates associated with dystonia. Exploiting fluorophore-derivatized MLF2 constructs, we developed a high-content platform and computational pipeline to screen modulators of NE condensates across chemical and genetic space. We identified RNF26 and ZNF335 as protective factors that prevent the buildup of nuclear condensates sequestering K48-linked polyubiquitinated proteins. Chemical screening identified four FDA-approved drugs that potently modulate condensates by resolving polyubiquitinated cargo and MLF2 accumulation. Our exploratory integrated chemical-genetics approach suggests that modulation of zinc, and potentially autophagy and oxidative stress, is critical for condensate modulation and nuclear proteostasis, offering potential therapeutic strategies for neurological disorders. Application of our platform to a genome-wide CRISPR KO screen identified strong enrichment of candidate genes linked to primary microcephaly and related neurodevelopmental disorders. Two hypomorphic microcephaly-associated alleles of ZNF335 failed to rescue nuclear condensate accumulation in ZNF335 KO cells, suggesting that aberrant condensates and impaired nuclear proteostasis may contribute to the pathogenesis of microcephaly. HIGHLIGHTS MLF2 emerges as a disease-agnostic condensate biomarker co-localizing with TDP-43 and G3BP1 FDA-approved drugs target condensates linked to perturbed proteostasis. RNF26 and ZNF335 are identified as modulators of nuclear phase transitions. Microcephaly patient disease alleles fail to counteract aberrant condensates.

The cell surface harbors thousands of distinct proteins whose function depend on continuous cycles of internalization and replenishment. Disturbances in this turnover are typical hallmarks of many diseases. Yet, tools to study the dynamics of most surface proteins are suboptimal or unavailable. Here, we present a new method that enables the analysis of surface protein turnover of virtually any surface protein at endogenous levels. Our approach combines CRISPR/cas9-mediated genome engineering with a cleavable recombinant probe, which addresses many of the shortcomings of current methodologies. We demonstrate the capabilities our method by studying the internalization behavior of a previously uncharacterized surface protein and by assessing the effect of ligands and activity modulators in the endocytic behavior of established receptors. In summary, our method represents a versatile strategy to explore surface protein biology and enhances our ability to study the mechanisms of membrane protein retrieval and recycling.

ABSTRACT Precise genome editing of induced pluripotent stem cells (iPSC) holds great promise for engineering advanced cell therapies. CRISPR-Cas systems have been widely adopted in genome engineering applications, however their dependence on genotoxic DNA double strand breaks (DSBs) presents challenges in hypersensitive iPSCs. Base editors are capable of both modifying and ablating gene function without generating DSBs making them an attractive solution for iPSC engineering. Here we report efficient and durable multiplexed target knockout with minimal impact on cell viability and expansion with a cytosine base editor composed of nCas9-UGI and Rat APOBEC1 assembled using the Pin-point™ platform (nCas9-UGI:rAPO). Minimal p53-mediated DNA damage signalling occurred independently of the number of simultaneous edits installed, and this could be further reduced by modulating the assembly of the base editor complex. Whereas non-homologous end-joining-mediated DNA damage repair led to p53-mediated selection against imprecise editing outcomes and an associated reduction in the on-target efficiency of multiplexed SpCas9 nuclease editing, p53 activity was dispensable for maintaining genome integrity during the base editing process. The Pin-point platform therefore enables the assembly of base editors optimised for high editing efficiency with substantially reduced risk of selecting for defective DNA damage responses inherent to DSB-dependent editing systems.