Adeno associated virus (AAV)-mediated delivery of CRISPR associated nucleases (AAV-CRISPR) is a promising solution to treat genetic diseases such as Duchenne Muscular Dystrophy (DMD) and is now in early clinical trials. However, genotoxicity and immunogenicity concerns have hindered clinical translation. Due to the complex etiology associated with DMD, the post-transduction consequences of double-stranded breaks induced by AAV-CRISPR in disease models are unclear. This barrier is partially conferred by conventional sequencing methods where common outcomes of AAV-CRISPR editing often escape detection. However, recent reports of novel long-read sequencing approaches permit comprehensive variant detection using a broader sequence context. Here, we comprehensively investigated genomic and transcriptomic post-AAV-CRISPR transduction consequences in myoblast cells and a DMD mouse model following intramuscular and intravenous AAV-CRISPR therapy using both long- and short-read sequencing techniques. Structural variant characterization indicates that unintended on-target large insertions and inversions are common editing outcomes. We demonstrate that combining adaptive sampling with nanopore Cas9-targeted sequencing (AS-nCATS) for long-read quantification of AAV integration is synergistic for detecting difficult-to-amplify editing events. This unbiased data suggests that full-length AAV integration is equally as probable as the on-target deletion. Further, we develop a Nanopore Rapid Amplification of cDNA Ends (nRACE-seq) pipeline for long-read detection of unknown 5’ or 3’ ends of edited transcripts. The nRACE-seq approach effectively detects the presence of AAV- Dmd chimeric transcripts, erroneous splicing events, and off-target AAV integration sites. In summary, our findings offer insights into the adaptation of AAV-CRISPR DSB-mediated therapeutics for monogenic diseases and promote the standardization of CRISPR evaluation. We highlight the importance of coupling polymerase-based and polymerase-free methods in long-read sequencing to assess editing outcomes as the field progresses toward clinical applications.

The transcription factor STAT3 plays broad roles in epithelial biology, yet its function in human esophageal development remains undefined. Using 2D and 3D human induced pluripotent stem cell (hiPSC)-derived platforms, we investigated how STAT3 regulates esophageal epithelial differentiation. We find that STAT3 is dispensable for definitive endoderm and anterior foregut endoderm specification but becomes essential during the transition to esophageal progenitor cells (EPCs). Inhibition of STAT3, via CRISPR-mediated knockout or siRNA, impairs the expression of key EPC and differentiation markers, including TP63 , and disrupts 3D organoid formation. These defects are accompanied by reduced epithelial proliferation. Notably, STAT3 is highly expressed in human fetal esophageal tissues and hiPSC-derived organoids, while its deletion in the developing mouse esophagus does not affect epithelial architecture, highlighting species-specific differences. Together, these findings identify STAT3 as a critical determinant of basal cell identity and epithelial morphogenesis, revealing a developmental checkpoint in early human esophageal lineage commitment.

The DNA-incorporating nucleoside analogs azacytidine (AZA) and decitabine (DEC) have clinical efficacy in blood cancers, yet the precise mechanism by which these agents kill cancer cells has remained unresolved -- specifically, whether their anti-tumor activity arises from conventional DNA damage or DNA hypomethylation via DNA methyltransferase 1 (DNMT1) inhibition. This incomplete mechanistic understanding has limited their broader therapeutic application, particularly in solid tumors, where early clinical trials showed limited efficacy. Here, through the assessment of drug sensitivity in over 600 human cancer models and comparison to a non-DNA-damaging DNMT1 inhibitor (GSK-3685032), we establish DNA hypomethylation, rather than DNA damage, as the primary killing mechanism of AZA and DEC across diverse cancer types. In further support of an epigenetic killing mechanism, CRISPR drug modifier screens identified a core set of chromatin regulators, most notably the histone deubiquitinase USP48, as AZA and DEC protective factors. We show that USP48 is recruited to newly hypomethylated CpG islands and deubiquitinates non-canonical histones, establishing USP48 as a key molecular link between the two components of epigenetic gene regulation: DNA methylation and chromatin modification. Furthermore, loss of USP48, which occurs naturally through biallelic deletions in human cancers, sensitized both hematologic and solid tumors to DNMT1 inhibition in vitro and in vivo. Our findings elucidate the epigenetic mechanism of action of AZA and DEC and identify a homeostatic link between DNA methylation and chromatin state, revealing new therapeutic opportunities for DNMT1 inhibitors in solid tumors.

Zonula occludens-1 (ZO-1), encoded by the TJP1 gene, is a crucial scaffolding protein within tight junctions that maintains epithelial and endothelial barrier integrity. In addition to its structural role, ZO-1 participates in signal transduction pathways that influence various cellular processes such as proliferation, differentiation, and apoptosis. Increasing evidence suggests that tight junction proteins, including ZO-1, play important regulatory roles in tumor progression, particularly by modulating metastasis, cell polarity, and vascular remodeling. Ovarian cancer, the most lethal gynecologic malignancy, is characterized by rapid growth, peritoneal dissemination, and a strong reliance on tumor angiogenesis. However, the specific role of ZO-1 in regulating angiogenesis within ovarian cancer remains poorly defined. In this study, we used CRISPR-Cas9-mediated gene editing to generate TJP1 knockout (KO) ovarian cancer cell lines and investigated the impact of ZO-1 loss on the expression of angiogenesis-related genes. Transcriptomic and qRT-PCR analyses revealed upregulation of KLF5 and IL-8, both of which are well-established pro-angiogenic factors. Furthermore, functional assessment using a Matrigel™ tube formation assay demonstrated that conditioned media from ZO-1-deficient cells significantly enhanced endothelial tube formation. These findings indicate that ZO-1 loss promotes a pro-angiogenic tumor microenvironment, likely through modulation of key signaling molecules such as KLF5 and IL-8. Therefore, ZO-1 may serve as a potential suppressor of angiogenesis and a therapeutic target in ovarian cancer.

ABSTRACT Embryonic development is precisely shaped by maternal and zygotic factors. These maternal factors exert their influence through maternal effects, a phenomenon where an offspring’s phenotype is determined, at least in part, by the mother’s environment and genotype. While environmental maternal effects can cause phenotypes that present both early and later in life, genetic maternal effects generally induce phenotypes in the earliest embryonic stages. Here, we reveal a genetic maternal effect that influences the development of cells that arise after early embryogenesis, highlighting that specific cell types can be susceptible to late-onset genetic maternal effects. Using zebrafish to study microglia, the resident immune cells of the brain, we identified a mutation in sry-related HMG box gene-17 (sox17) that exhibits a maternal effect phenotype that presents as a reduction of microglia in the brain and precursors in the yolk sac. We demonstrate that sox17 is expressed in microglia and their yolk sac precursors and is maternally-loaded. We show that sox17 restoration via embryonic injection reverses the maternal effect on microglia and yolk sac cells in sox17 mutants. To identify additional genes interacting with sox17 , we nominated genes from scRNA sequencing analysis of mouse embryonic microglia to perform a genetic screen using CRISPR mutagenesis and a custom-built robot that captures confocal images of the zebrafish brain in high-throughput. This screen identified f11r.1, gas6, and mpp1 as modifiers of microglia abundance in the embryonic brain, which we demonstrated are also expressed in zebrafish microglia. Transcriptional and mutant analyses with these new modifiers suggest that sox17 positively regulates mpp1 transcription. These results demonstrate that microglia are susceptible to genetic maternal effects, in addition to their known sensitivity to environmental maternal effects. Our findings reveal a late-onset phenotype associated with the maternal genotype, expanding the recognized impact of genetic maternal effects beyond initial embryo viability and into long-term vigor.

Pathogenic KCNQ2 variants are associated with developmental and epileptic encephalopathy (KCNQ2-DEE), a devastating disorder characterized by neonatal-onset seizures and impaired neurodevelopment with no effective treatments. KCNQ2 encodes the voltage-gated potassium channel K V 7.2, which regulates action potential threshold and repolarization. However, the relationship between K V 7.2 dysfunction and abnormal neuronal activity remains unclear. Here, we use human induced pluripotent stem (iPSC)-derived neurons from 5 KCNQ2-DEE patients with pathogenic variants and CRISPR/Cas9-corrected isogenic controls to investigate pathophysiological mechanisms. We identify a common dyshomeostatic enhancement of Ca 2+ -activated small conductance potassium (SK) channels, which drives larger post-burst afterhyperpolarizations in KCNQ2-DEE neurons. Using microelectrode arrays (MEAs), we recorded over 18 million extracellular spikes from >8,000 neurons during 5 weeks in culture and then applied supervised and unsupervised machine learning algorithms to dissect time-dependent functional neuronal phenotypes that defined both patient-specific and shared firing features among KCNQ2-DEE patients. Our analysis identified irregular spike timing and enhanced bursting as functional biomarkers of KCNQ2-DEE and demonstrated the significant influence of genetic background on phenotypic diversity. Importantly, using unbiased machine learning models, we showed that chronic treatment with the K V 7 activator retigabine rescues the disease-associated functional phenotypes with variable efficacy. Our findings highlight SK channel upregulation as a critical pathophysiological mechanism underlying KCNQ2-DEE and provide a robust MEA-based machine learning platform useful for deciphering phenotypic diversity amongst patients, discovering functional disease biomarkers, and evaluating precision medicine interventions in personalized iPSC neuronal models.

TGF-β-mediated signaling controls mast cell (MC) development and exerts anti-inflammatory functions, while antigen/allergen (Ag)-triggered FcϵRI activation commands pro-inflammatory reactions. TGF-β induces strong C-terminal and low linker phosphorylation of SMAD2. In contrast, Ag triggers immediate, MEK-dependent SMAD2 linker phosphorylation only. Both stimuli can positively or negatively influence each others effects on MC activation in a gene-dependent manner. However, the molecular and cellular mechanisms of SMAD2 in MCs still need to be elucidated. To decipher the role(s) of SMAD2 in MCs, SMAD2 was ablated in PMC-306 MCs using CRISPR/Cas9, and the effects were studied after TGF-β and/or Ag stimulation. The absence of SMAD2 led to increased proliferation and survival, as well as decreased transcription of target genes like Smad7 and Jun in steady state and after TGF-β treatment. Interestingly, SMAD2 was found to regulate the strength and kinetics of TGF-β-mediated SMAD1/5 activation, resulting in augmented expression of genes like Id2 and Id3 in SMAD2-deficient MCs. Unexpectedly, SMAD2 was observed to license Ag-triggered production of pro-inflammatory cytokines, such as IL-6 and TNF, by monitoring expression of secondary repressive signaling elements. Re-introducing SMAD2 restored these events with varying sensitivity depending on the receptor system triggered. Our findings reveal SMAD2 as an initial hub in TGF-β-SMAD1/5 and Ag-FcϵRI signaling, offering new possibilities for therapeutic intervention in both TGF-β-controlled and Ag-triggered MC functions using potential SMAD2 activators or inhibitors.

Trafficking from the endoplasmic reticulum to the Golgi apparatus comprises the first steps toward the correct localization of 30% of eukaryotic proteins. Coat protein complexes COPII and COPI are involved in forward and retrograde transport of cargo and cargo receptors between the ER and the Golgi. Although COPII forms coated vesicles in vitro, the biogenesis, morphology and organization of transport carriers in mammalian cells is debated. We use in situ cryo-electron tomography and super-resolution fluorescence microscopy to reveal the molecular architecture of ER exit sites in human cells. We visualise ribosome-exclusion zones enriched with COPII and COPI-coated vesicles and thus resolve the debate regarding the existence of COPII coated vesicles. COPII vesicles derive from ER membranes, whereas COPI vesicles originate from the ER-Golgi intermediate compartment. We quantify coated vesicle morphology and positioning with respect to other ER exit site components, providing a molecular description of the mammalian early secretory pathway.

CRISPR-Cas12a is a programmable, RNA-guided endonuclease that has revolutionized biotechnology, with applications in genome engineering and diagnostics. To induce nuclease activity, Cas12a must first interact with the target dsDNA duplex by associating with a short protospacer adjacent motif (PAM) in the sequence. In this study we have split this target duplex to create PAM-proximal and PAM-distal duplex regions, which has allowed us to regulate trans-cleavage activity when these regions are included in combination or separately. These observations on Cas12a activity led to hypotheses into the related functional mechanisms, which we have tested and that have highlighted DNA/protein interactions during Cas12a complex assembly that were not otherwise apparent. Selective destabilization of the nucleic acid complexes appears to drive greater reliance on the Cas12a protein for complex stability. We have exploited this to provide significant improvements in both structural selectivity and nucleotide specificity in PAM-proximal and PAM-distal duplex regions, respectively. The result is an architecture that shows promise as a PAM-free ultra-specific platform to resolve single nucleotide polymorphisms.

Shikonin, a 1,4-naphthoquinone derivative produced by several Boraginaceae species, exhibits unique pharmacological properties and is used as a natural dye. The regulatory factors of shikonin production have been demonstrated using a cell culture system of Lithospermum erythrorhizon . Among these factors, copper is known to be the strongest enhancer of shikonin production. Although shikonin biosynthesis has been studied for over 40 years, the steps of naphthalene ring formation are still unknown, as is the reason for the effect of copper. In this study, we explored candidate genes associated with shikonin production using a PCR-select subtraction experiment. Polyphenol oxidase (PPO), a dicopper-dependent oxidoreductase, was highlighted because it showed synchronous expression with shikonin production. Transcriptome analysis of hairy roots and cultured cells of this plant revealed that, of the five PPO genes expressed in L. erythrorhizon , only PPO1 showed a strong correlation with shikonin production. Next, we generated genome-edited hairy roots of LePPO1 using CRISPR/Cas9-mediated mutagenesis to analyze its impact on shikonin derivative and other specialized metabolite production. The results showed that shikonin content was markedly reduced in all LePPO1 -ge lines. Interestingly, the content of deoxyshikonofuran, a hydroquinone derivative and shunt product that branches after GHQ-3′′-OH in the shikonin biosynthetic pathway, remained unaffected in the LePPO1 -ge lines. These findings suggest that LePPO1 participates in naphthalene ring formation and explain why a copper ion is crucial for shikonin biosynthesis.

ABSTRACT Down Syndrome (DS) is the most abundant genetic form of mental retardation. It is caused by the triplication of partial or complete human chromosome 21 (HSA21). The molecular mechanisms causing it are not fully understood. Previous studies identified “Down syndrome Critical Region” (DSCR) genes that are essential or sufficient for the development of DS. However, these studies are largely inconclusive, due, in part, to the reliance on a small number of epidemiological cases. Amyloid precursor protein ( APP ) resides on HSA21 and is triplicated in DS. APP plays a role in developmental and post-natal neurogenesis, but is not thought to be part of the DSCR. The role of APP overdose in cortical malformation and cognitive impairments in DS is unknown. Mutations in APP cause familial Alzheimer’s disease (FAD). However, whether APP overdose is sufficient for the development of Alzheimer’s disease (AD) in DS is not fully understood. Here, we addressed the role of APP overdose in neuronal development and AD pathology. Using CRISPR/Cas9 gene editing, we eliminated one copy of APP from Down Syndrome-derived induced iPSCs DS APP(+/+/-) and examined the effect on neurogenesis, AD-related pathology and the expression levels of genes on HSA21 that are implicated in DS, neurodegeneration and inflammation.

Gene knock-in therapy has the potential to cure inherited liver diseases but is limited by low efficiency and delivery complexity. Here, we developed a single adeno-associated virus (AAV) vector system comprising a compact CRISPR effector, enAsCas12f, a guide RNA, and a donor template to enable therapeutic genome editing via non-homologous end joining (NHEJ). We targeted the system to the murine Alb locus and applied it to mouse models of hemophilia B, protein C (PC) deficiency, and ornithine transcarbamylase (OTC) deficiency. NHEJ-mediated knock-in showed higher efficiency than homology-directed repair, with successful therapeutic gene insertion in both neonatal and adult mice. The strategy restored plasma factor IX activity in hemophilia B ( F9 −/− ) mice, prolonged survival of PC-deficient ( Proc −/− ) mice, and prevented hyperammonemia and weight loss in OTC-deficient ( Otc spf-ash ) mice upon high protein challenge. Importantly, gene integration was restricted to the liver, with no evidence of germline transmission. This compact, all-in-one AAV knock-in platform simplifies vector production, enables efficient delivery, and achieves reliable transgene expression in vivo . Our findings highlight the potential of liver-targeted knock-in genome editing as a transplant-independent treatment for neonatal-onset metabolic diseases, offering a clinically feasible path towards curative gene therapies for a wide range of monogenic liver disorders.

Summary Background 4-1BB (CD137), a member of the TNF receptor superfamily, is a critical co-stimulatory receptor for CD8⁺ T cell activation and regulatory T cell (Treg) expansion. While its ligand 4-1BBL is typically expressed by professional antigen-presenting cells, several carcinomas also express 4-1BBL, though its function in the tumor microenvironment remains poorly defined. Methods We analyzed 4-1BBL expression across human tumors and found papillary renal cell carcinoma (pRCC) to exhibit the highest levels. Using The Cancer Genome Atlas, we found high 4-1BBL expression correlated with poor overall survival in pRCC. To study its role in vivo, we established an orthotopic humanized mouse model of pRCC by grafting ACHN cells into the renal capsule of mice reconstituted with human CD34⁺ hematopoietic stem cells. We then performed CRISPR-mediated deletion of 4-1BBL in tumor cells, followed by flow cytometry and single-cell RNA sequencing of tumor-infiltrating immune cells. Results Loss of tumor-derived 4-1BBL resulted in accelerated tumor growth and decreased immune cell clustering. In the absence of 4-1BBL, CD8⁺ T cells displayed elevated expression of PD-1, TIM-3, LAG-3, granzyme B, perforin, and NKG7, indicating a cytotoxic yet exhausted phenotype. Treg were only modestly impacted. Tumor-infiltrating CD8⁺ T cells expressed high levels of 4-1BBL and showed transcriptional signatures of altered AP-1 factors and enhanced PI3K pathway signaling. Conclusions Our findings uncover a previously unrecognized role for tumor- and T cell–derived 4-1BBL in sustaining cytotoxic CD8⁺ T cell functionality and limiting their exhaustion. This reveals a potential immune-regulatory axis that could be exploited for therapeutic modulation in renal cell carcinoma.

Johanson-Blizzard Syndrome (JBS) is an autosomal recessive spectrum disorder associated with the UBR-1 ubiquitin ligase that features developmental delay including motor abnormalities. Here, we demonstrate that C. elegans UBR-1 regulates high-intensity locomotor behavior and developmental viability via both ubiquitin ligase and scaffolding mechanisms. Super-resolution imaging with CRISPR-engineered UBR-1 and genetic results demonstrated that UBR-1 is expressed and functions in the nervous system including in pre-motor interneurons. To decipher mechanisms of UBR-1 function, we deployed CRISPR-based proteomics using C. elegans which identified a cadre of glutamate metabolic enzymes physically associated with UBR-1 including GLN-3, GOT-2.2, GFAT-1 and GDH-1. Similar to UBR-1, all four glutamate enzymes are genetically linked to human developmental and neurological deficits. Proteomics, multi-gene interaction studies, and pharmacological findings indicated that UBR-1, GLN-3 and GOT-2.2 form a signaling axis that regulates glutamate homeostasis. Developmentally, UBR-1 is expressed in embryos and functions with GLN-3 to regulate viability. Overall, our results suggest UBR-1 is an enzyme hub in a GOT-2.2/UBR-1/GLN-3 axis that maintains glutamate homeostasis required for efficient locomotion and organismal viability. Given the prominent role of glutamate within and outside the nervous system, the UBR-1 glutamate homeostatic network we have identified could contribute to JBS etiology.

Toxoplasma and other Apicomplexan parasites, switch between different developmental stages to persist in and transmit between hosts. Toxoplasma can alternate between systemic tachyzoites and encysted bradyzoite forms found in the CNS and muscle tissues. How parasites sense these tissue types and trigger differentiation remains largely unknown. We show that Toxoplasma differentiation is induced under glucose-limiting conditions and using a CRISPR screen identify parasite genes required for growth under these conditions. From ∼25 identified genes important for differentiation we show that lactate and glutamine metabolism is linked to differentiation and demonstrate the importance of an E3 ubiquitin ligase complex, orthologous to glucose induced degradation deficient (GID) complex in yeast and CTLH complex in humans. We show that TgGID likely regulates translational repression of a key transcription factor required for differentiation, BFD1, through its 3’ utr. Overall, this work provides important new insight into how these divergent parasites sense different host cell niches and trigger stage conversion through a ubiquitination-dependent program.

Abstract It was recently shown that inhibition of polo-like kinase 4 (PLK4) induces TP53 -dependent synthetic lethality in cancers with chromosome 17q-encoded TRIM37 copy number gain due to cooperative regulation of centriole duplication and mitotic spindle nucleation. We show here that chromosome 17q/TRIM37 gain is a pathognomonic feature of high-risk neuroblastoma and renders patient-derived cell lines hypersensitive to the novel PLK4 inhibitor RP-1664. We demonstrate that centriole amplification at low doses of RP-1664 contributes to this sensitivity in a TRIM37 - and TP53 -independent fashion. CRISPR screens and live cell imaging reveal that upon centriole amplification, neuroblastoma cells succumb to multipolar mitoses due to an inability to cluster or inactivate supernumerary centrosomes. RP-1664 showed robust anti-tumor activity in 14/15 neuroblastoma xenograft models and significantly extended survival in a transgenic murine neuroblastoma model. These data support biomarker-directed clinical development of PLK4 inhibitors for high-risk neuroblastoma and other cancers with somatically acquired TRIM37 overexpression.

Abstract Despite initial responses, most patients with metastatic lung cancer—including those with EGFR mutations—ultimately develop resistance to targeted therapies. To systematically uncover mechanisms underlying this resistance, genome-wide CRISPR knockout and activation screens were conducted in EGFR-mutant lung cancer cell lines treated with EGFR inhibitors such as osimertinib and gefitinib. These screens highlighted a recurrent involvement of genes associated with the Hippo signaling pathway. Notably, a subset of tumor cells, termed 'persister' cells, survive initial osimertinib exposure by engaging non-genetic, transcriptional adaptation mechanisms that promote drug tolerance. Our studies, integrating both genetic and pharmacological approaches, identified Hippo pathway activation as a key driver of this drug-tolerant state. Importantly, co-inhibition of EGFR and the Hippo signaling axis led to a pronounced reduction in cell viability in both established cell lines and patient-derived organoids. These findings propose that dual targeting of EGFR and Hippo signaling may offer a promising therapeutic approach to overcome resistance in EGFR-mutant lung cancer.

Objectives: To explore the molecular chemistry and structural biology of bacteriophages as precision-guided therapeutic agents. This review reframes phages as programmable nanomachines governed by defined chemical interactions, focusing on their relevance to antimicrobial resistance, targeted lysis, and synthetic modification. Materials and Methods: A systematic review was conducted using recent peer-reviewed literature from PubMed, Scopus, and Google Scholar, covering bacteriophage structural biology, enzymatic lysis mechanisms, chemical modifications, genomic annotation, and bioengineering techniques. Studies were selected based on relevance to molecular chemistry, nanotechnology, and therapeutic development. Results: Bacteriophages demonstrate ligand-specific host recognition, capsid-mediated genome packaging, and enzymatic lysis using holins and endolysins. Advances in PEGylation, surface conjugation, and CRISPR engineering have expanded their therapeutic potential. Genomic tools now enable personalized phage matching, while hybrid phage-nanoparticle systems enhance targeting and delivery. Conclusions: Phages can be rationally designed as chemically programmable antimicrobials. Their structure–function relationship, enzymatic precision, and genomic adaptability position them as promising agents in the fight against multidrug-resistant pathogens. Integration of chemistry, bioinformatics, and synthetic biology enables development of next-generation phage therapeutics.

Abstract Functional genomics has been hampered by the paucity of efficient methods that connect genotype and metabolic phenotype at single-cell resolution. Using the industrial microalga Nannochloropsis oceanica as a model, we introduced a platform that comprises a genome-wide single-gene-edited mutant library and high-throughput Raman-activated Cell Sorting (RACS). The CRISPR/Cas-generated library consists of 3,567 microalgal mutants derived from 2,397 effective guide RNAs. Label-free sorting of the library for high carotenoid content by RACS unravels mutations in the violaxanthin de-epoxidase ( noVDE ) or in the proteasome assembly chaperone 4 ( noPAC4 ) genes. Knocking out all five known noVDE s reveal that the high carotenoid content is due to violaxanthin increase, whilst noPAC4 knockout boosted carotenoid content with elevations in violaxanthin, zeaxanthin, and β-carotene. Genetic and transcriptomic evidences suggest two previously unknown modes of carotenogenesis regulation mediated by noPAC4: epigenetic mechanisms via histone deacetylase (HDAC) and post-translational controls by the 26S proteasome. Therefore, by label-freely sorting single-cell metabolic phenotype and rapidly yet unambiguously tracing it to a genotype, this new forward-genetics approach can greatly accelerate the discovery of new genes and pathways.

Abstract Cancer therapy faces a critical need for treatments that selectively eliminate malignant cells without harming healthy tissue. The Proteus Project addresses this challenge by engineering a programmable gene circuit that couples CRISPR-based RNA sensing with an inducible cell death mechanism. Specifically, we repurpose a novel Type III-E CRISPR-Cas system (“Craspase”) – an RNA-guided protease complex – to detect cancer-specific RNA transcripts and, in response, cleave engineered gasdermin fusion proteins to trigger cell death (1)(2). We constructed the Proteus system in Saccharomyces cerevisiae as a surrogate model, integrating components that sense an oncogenic KRAS mutation and execute targeted pyroptosis (inflammatory apoptosis-like cell death). Preliminary results demonstrate successful assembly of the Craspase– gasdermin circuit, expression of key proteins, and proof-of-concept cell killing specifically in the presence of the oncogenic RNA trigger. This work, conducted as part of the iGEM 2025 SynBio Collective, showcases a modular synthetic biology approach for precision oncology therapeutics. The ongoing study underscores the potential of CRISPR-guided proteases in in situ cancer cell ablation and sets the stage for future validation in mammalian systems.

ABSTRACT CTCF-mediated chromatin loops are known to influence gene regulation, yet their role in pre-mRNA splicing remains incompletely understood. Here, we demonstrate that structural variants (SVs) at the anchors of intronic CTCF loops can modulate exon usage. By integrating high-resolution three-dimensional (3D) genome organization and gene expression datasets from C57BL/6J (B6) and 129S1/SvImJ (129S) mouse embryonic stem cells (ESCs), with structural variant (SV) maps from the 129S mouse, we identified thousands of intron-anchored CTCF loops. Our data indicate that SVs intersecting loop anchors are more frequently associated with differential exon inclusion events than with changes in overall gene expression. CRISPR/Cas9 deletion of two SV-harboring intronic CTCF sites in Numbl and Ireb2 validated the predicted splicing shifts observed between B6 and 129S ESC that correspond with diminished long-range chromatin looping. Our findings reveal a direct mechanistic link between 3D genome architecture and alternative splicing and highlight non-coding SVs as modulators of transcript diversity. Our study has thus identified a novel class of CTCF-bound regulatory elements regulating alternative splicing. Cataloging and validating these functional elements will elucidate molecular mechanisms underlying phenotypic variation within populations.

CHARGE syndrome is a developmental disorder that affects 1 in 10,000 births, and patients exhibit both physical and behavioral characteristics. De novo mutations in CHD7 (chromodomain helicase DNA binding protein 7) cause 67% of CHARGE syndrome cases. CHD7 is a DNA-binding chromatin remodeler with thousands of predicted binding sites in the genome, making it challenging to define molecular pathways linking loss of CHD7 to CHARGE phenotypes. To address this problem, here we used a previously characterized zebrafish CHARGE model to generate transcriptomic and proteomic datasets from larval zebrafish head tissue at two developmental time points. By integrating these datasets with differential expression, pathway, and upstream regulator analyses, we identified multiple consistently dysregulated pathways and defined a set of candidate genes that link loss of chd7 with disease-related phenotypes. Finally, to functionally validate the roles of these genes, CRISPR/Cas9-mediated knockdown of capgb , nefla , or rdh5 phenocopies behavioral defects seen in chd7 mutants. Our data provide a resource for further investigation of molecular mediators of CHD7 and a template to reveal functionally relevant therapeutic targets to alleviate specific aspects of CHARGE syndrome. Summary Statement We have identified Chd7 target genes capgb , nefla , and rdh5 that mediate CHARGE model phenotypes from transcriptomic and proteomic analysis of chd7 wild type, heterozygous, and homozygous mutant zebrafish brain tissue at two developmental time points.

Chimeric antigen receptor (CAR) T-cell therapy has transformed the treatment of hematologic cancers. However, its efficacy in solid tumors, including pancreatic ductal adenocarcinoma (PDAC), has been limited. By integrating modular CRISPR screening with immunocompetent orthotopic models of PDAC, we identified unknown tumor-intrinsic modulators of CAR T-cell therapy response. Disruption of genes involved in oxidative and proteotoxic stress, particularly the Nrf2 target Slc33a1 , sensitizes PDAC tumors to CAR T-cell killing. Single cell gene expression analyses revealed that CAR-T resistant tumors exhibit reduced Nrf2 pathway activity. Mechanistically, we show that Nrf2 pathway hyperactivation by genetic ablation of Keap1 or expression of a tumor-derived Keap1 allele sensitized PDAC tumors to CAR T-cell therapy. Thus, cell-intrinsic molecular states accompanying malignant progression can sensitize tumor cells to cell-based immunotherapies. These molecular mechanisms could be exploited to augment both the efficacy of CAR-T cell therapy in solid malignancies, and may allow patient stratification by tumor genotype. Statement of significance CAR T-cell therapy remains an unsolved challenge for pancreatic cancer. The discovery of tumor-intrinsic mechanisms of resistance has been largely limited by current experimental models. Using large-scale genomic screening in an orthotopic, immunocompetent model of pancreatic cancer, we uncover a role for cell-intrinsic metabolic states in regulating CAR T-cell response.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterised by motor neuron deterioration. Genetic factors play a significant role in all cases, with 15 genome-wide significant study (GWAS) risk loci identified to date. Follow-up of these loci is a powerful strategy for research translation, as drug targets supported by genetic evidence are more likely to succeed in clinical development. Here, we focus on the RPSA-MOBP locus on chromosome 3 (lead SNP, rs631312, OR = 1.08 95% CI: 1.06–1.10, p = 3.3 × 10⁻¹²). We employ integrative in silico analyses to prioritise candidate genes, combining multiple ‘omics-based approaches, including Functional Mapping and Annotation (FUMA), Polygenic Priority Scoring (PoPS), Transcriptome-Wide Association across/within tissues (TWAS), gene-based test (mBAT-combo), chromatin interaction mapping (H-MAGMA), and Summary data Mendelian Randomisation (SMR), with GWAS data ( N cases = 29,612, N controls = 122,656). Both RPSA and MOBP were prioritised as candidate genes in multiple analyses. In-vivo expression analyses in ALS blood or iPSC-motor neurons were unremarkable for these genes but also other-relevant ALS genes. RPSA , highly conserved in zebrafish (92% homology), was selected for functional modelling, noting previously generated Mobp -ko mice show minimal phenotypic changes. CRISPR/Cas9-induced rpsa loss-of-function (LOF) in zebrafish triggers progressive and severe phenotypes mimicking pathology observed in SMN- and TDP43-deficient zebrafish, two key proteins/genes associated with diseases of the motor neurons. RPSA -deficient animals exhibit marked motor neuron axon pathology, progressive loss of motor function and rapid decline culminating with premature death at around 7 days- post-fertilisation. These phenotypes were notably similar to those observed in SMN and TDP-43 zebrafish models, together with prominent cardiovascular abnormalities. This study identifies RPSA as a critical gene for motor neuron health, with implications for ALS pathogenesis. The RPSA/MOBP locus is also associated with other neurodegenerative diseases including frontotemporal dementia/FTD, corticobasal degeneration/CBD and progressive supranuclear palsy/PSP, highlighting its potential as a therapeutic target for multiple conditions.

TRIB members (TRIB1, TRIB2 and TRIB3) represent atypical members of the serine/threonine kinase superfamily and are involved in multiple biological processes such as cell proliferation and differentiation. TRIB roles in GC are not fully investigated. We hypothesized that TRIB members play crucial roles in regulating GC activity and may activate separate signaling pathways. TRIB1 and TRIB3 are induced in GC of ovulatory follicles (OF) following hCG injection as compared to dominant follicles (DF) whereas TRIB2 is suppressed by hCG in OF. Protein analyses of cultured primary GC showed that luteinizing hormone (LH) treatment inhibited TRIB2 and induced TRIB3, while follicle-stimulating hormone (FSH) treatment increased TRIB2 and TRIB3 expression but at different times. These results demonstrate a different regulation of TRIB members during follicular development and in response to gonadotropins. TRIB3 inhibition via CRISPR/Cas9 showed a positive effect on AKT signaling pathway while showing negative effects on P38 MAPK signaling. Overall, TRIBs may play crucial roles in regulating GC function and activity and may activate separate signaling pathways, which impact follicular development, ovulation and luteinization.