Biology subjects

Zhu, R.

Publications and source records attributed to Zhu, R..

At least 19 recordsLinked to original sources

bioRxiv2026

Fine-tuning an intracellular allosteric pocket programs GPCR signaling bias

Biased signaling of G protein-coupled receptors (GPCRs) offers a route for precise therapeutics with fewer adverse effects. Intracellular allosteric ligands can impose such bias, yet how subtle chemical changes within these pockets reshape receptor-transducer coupling and downstream signaling remains unclear. Here, we systematically fine-tuned the intracellular allosteric pocket of neurotensin receptor 1 (NTSR1) through chemical modification of the {beta}-arrestin-biased ligand SBI-553. Minor changes to the ligand scaffold altered transducer-coupling preferences, generating distinct profiles of G-protein and {beta}-arrestin signaling. Cryo-electron microscopy structures of NTSR1-{beta}-arrestin2 complexes bound to designed allosteric probes revealed ligand-dependent remodeling of the intracellular cavity that reshapes the receptor-effector interface. Alanine scanning identified transducer-sensitive residues within and beyond the immediate ligand-contact region. In mice, SBI-553 and N065 produced distinct physiological profiles, with N065 improving survival in a lethal endotoxemia model. Finally, phosphoproteomic profiling revealed partially overlapping but distinct downstream signaling profiles among the three modulators. Together, these findings establish the intracellular allosteric pocket as a tunable determinant of transducer coupling and show that closely related modulators can generate distinct signaling and physiological profiles.

pharmacology and toxicology↗

bioRxiv2026

Longitudinal Metaproteomic and Lysine Crotonylation Remodeling Defines Clinical Response to Fecal Microbiota Transplantation in Ulcerative Colitis

Background Fecal microbiota transplantation (FMT) can induce steroid-free clinical remission in a subset of patients with ulcerative colitis (UC), but treatment responses remain heterogeneous and are not fully explained by taxonomic microbiome shifts. To define functional molecular features associated with FMT efficacy, we performed integrated longitudinal fecal metaproteomic and lysine crotonylation (Kcr) profiling. Methods We analyzed 87 fecal samples from 23 patients with active UC collected at baseline and at weeks 1, 8, and 12 after FMT. Patients were classified as responders (n = 13) or non-responders (n = 10) according to steroid-free clinical remission at week 12. Across samples, we identified an average of 3,682 proteins and 562 Kcr sites per sample. Results Responders showed greater metaproteomic and meta-crotonylomic complexity at week 12, sustained enrichment of Prevotella and Faecalibacterium, and eight directionally consistent baseline-altered proteins with candidate prognostic value. After FMT, responders exhibited coordinated microbial functional remodeling, including increased proteins involved in short-chain fatty acid (SCFA) metabolism, acyl-CoA-centered pathways, nutrient transport, and polysaccharide degradation, together with attenuation of host neutrophil-related inflammatory protein and Kcr signatures. Non-responders showed minimal or opposing molecular changes. Conclusions: These findings nominate longitudinal fecal metaproteomic and Kcr remodeling as functional molecular readouts of clinical response to FMT in UC and highlight lysine crotonylation as an underexplored metabolite-sensitive layer linking microbial metabolic restoration with host inflammatory resolution.

microbiology↗

bioRxiv2026

Hypothermic and Normothermic Machine Perfusion Differentially Shape the Renal Cellular Landscape

The shortage of donor organs has led to an increased use of extended-criteria donors and donation after circulatory death for kidney transplantation, both of which are associated with graft injury. Although machine perfusion (MP) offers a preservation strategy, the most beneficial approach for kidneys with respect to effective graft preservation remains unclear. We compared oxygenated hypothermic (HMP) and normothermic machine perfusion (NMP) using a murine kidney perfusion model regarding intra-graft immune cell composition, viability, and inflammatory responses. NMP resulted in more severe tubular injury, accompanied by increased immune cell mobilization out of the graft. NMP increased early apoptosis among kidney-resident immune-cell populations, whereas endothelial cells showed increased late apoptosis after both strategies. Proximal tubular-cell apoptosis remained comparable to naive tissue, and neither modality reduced reactive oxygen species levels. Both protocols induced antioxidant and hypoxia-responsive genes compared with static cold storage, while NMP elicited a stronger inflammatory response. Treatment with the senolytic ABT-263 demonstrated the models feasibility for investigating renal injury, cellular viability, and immune activation during MP.

immunology↗

bioRxiv2026

Antiviral Sulfonated Polymersomes Inhibit Corona-, Influenza-, and Alphavirus

A broad-spectrum antiviral is required to enable rapid intervention against current, newly emerging and mutating viruses. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus (IAV), Sindbis virus (SINV) and chikungunya virus (CHIKV) all engage negatively charged moieties on host cells, such as heparan sulfate (HS) and sialic acid, to facilitate attachment for entry and infection. We designed HS-mimetic nanoparticles in the form of polymer vesicles (polymersomes) by blending membrane-forming and sulfonated copolymers. These biocompatible polymersomes successfully inhibited host cell infection of HS-dependent SARS-CoV-2 and HS-independent IAV in vitro with sub-nanomolar potency. Multivalent interaction of several virus ligands with a single polymersome as quantified by fluorescence correlation spectroscopy likely contributes to their potency. The sulfonated polymersomes also functioned in vivo as demonstrated by a reduction in the spread of SINV, an alphavirus model for CHIKV, in zebrafish embryos, which led to a significantly increased survival. Hence, our polymersomes could form the basis for an urgently required multifunctional broad-spectrum antiviral nanomedicine.

bioengineering↗

bioRxiv2026

Variant-Specific Rewiring of Lysosomal Positioning by STARD9 Produces Opposite Cholesterol Trafficking Outcomes

Residual cardiovascular risk persists despite lipid-lowering therapy, independent of LDL and Lp(a), reflecting cholesterol dysregulation. We identify STARD9, a lysosomal cholesterol-sensing kinesin, as a causal gene for divergent familial dyslipidemias and premature atherosclerosis. Its START domain binds cholesterol to govern lysosomal positioning. Rare variants segregate with autosomal dominant hypercholesterolemia and premature coronary disease, while common variants associate with reduced HDL and elevated triglycerides. Both converge on lysosomal cholesterol sequestration, ER depletion, mTORC1 SREBP2 activation, impaired autophagy, and NF-kB inflammation, yet diverge through opposite lysosomal positioning: the rare variant disperses lysosomes peripherally, phenocopying START domain loss across cholesterol sequestration, lysosomal positioning, and TFEB activation, triggering LDLR degradation and CASM/TFEB-dependent efflux that preserves HDL, whereas the common variant causes perinuclear retention that suppresses TFEB dependent lipid-handling transcripts, yielding hypertriglyceridemia and low HDL. In Stard9-knockout mice, enterocyte cholesterol sequestration drives SREBP2 dependent NPC1L1 upregulation and apical localization, increasing intestinal cholesterol absorption. These findings position lysosomal cholesterol trafficking as a targetable node in statin-refractory cardiovascular disease.

genetics↗

bioRxiv2026

M.FcoHsdM-dependent DNA methylation coordinates secretion-associated gene expression, interbacterial competition, and virulence in Flavobacterium columnare

Flavobacterium columnare causes columnaris disease in freshwater fish, but the contribution of DNA methylation to its physiology and pathogenicity remains poorly understood. Here, we characterized M. FcoHsdM, a type I restriction-modification methyltransferase previously identified by nanopore methylome analysis. Deletion of the gene encoding M. FcoHsdM abolished methylation at its associated recognition motifs without affecting planktonic growth. The mutant exhibited impaired biofilm formation, gliding motility, colony spreading, and gelatinolytic activity, together with increased outer-membrane vesicle production and complete attenuation in a zebrafish infection model. Transcriptomic and extracellular proteomic analyses revealed broad changes in secretion and cell surface associated functions. In particular, genes encoding components of a putative type VI secretion system were coordinately downregulated, and the mutant showed reduced antagonistic activity against Aeromonas hydrophila. Several predicted type IX secretion system substrates and extracellular enzymes were also reduced, potentially contributing to the motility and virulence defects. Two such motifs were identified in tssP, which encodes a predicted Bacteroidota-specific type VI secretion system membrane-complex component. One motif overlapped a computationally predicted binding site for the leucine-responsive regulatory protein Lrp, raising the hypothesis that intragenic methylation may influence regulator occupancy at the tssP locus. Collectively, these findings identify M.FcoHsdM-dependent methylation as an important contributor to secretion-associated gene expression, interbacterial antagonism, and virulence in F. columnare.

microbiology↗

bioRxiv2026

Isolation, identification, and comparative genomic analysis of Vibrio harveyi as a causative agent for a skin ulcer disease of endangered fish Chinese bahaba (Bahaba taipingensis)

Chinese bahaba (Bahaba taipingensis) is a fish species endemic to China and listed as a Class I nationally protected wild animal. Although significant breakthroughs have been achieved in its artificial breeding, high mortality rates persist during cultivation. Beginning in August 2024, sustained mass mortalities occurred in artificially farmed Chinese bahaba at a farm in Guangdong Province. Diseased fish exhibited pronounced skin ulcers, ascites, gastroenteritis, and splenomegaly. Histopathological examination revealed severe necrotizing gastroenteritis, gill necrosis and edema, muscle congestion with inflammatory cell infiltration, hepatic congestion with marked vasculitis, renal interstitial necrosis with tubular atrophy, and abundant melanomacrophage centers in the spleen. Based on biochemical characterization and whole-genome phylogenomic analysis, the isolated strain HCY1 was identified as Vibrio harveyi. Artificial infection experiments with this strain resulted in 100% mortality and reproduced clinical signs similar to those in naturally infected fish. Antimicrobial susceptibility testing showed that HCY1 remained susceptible to florfenicol and trimethoprim-sulfamethoxazole, but was resistant to multiple antibiotics, including {beta}-lactams and tetracyclines. Comparative genomic analysis revealed that HCY1 carries various antimicrobial resistance genes primarily conferring resistance to {beta}-lactam and tetracycline antibiotics, consistent with the phenotypic resistance profile. The strain also harbored numerous virulence genes, including complete type III and type VI secretion systems (T3SS and T6SS), tlh, hap/vvp, and rtxB. Its plasmids encoded multiple virulence-enhancing genes, such as mcpA, pal, and esiB. To our knowledge, this is the first systematic investigation of a disease outbreak in cultured Chinese bahaba, and provides valuable insights for disease control and population conservation of this endangered species.

microbiology↗

bioRxiv2026

Efficacy of Flavobacterium spp. Vaccines in fish: A systematic review and meta-analysis

Flavobacterium species cause substantial economic losses in aquaculture, but no quantitative synthesis of vaccine efficacy across species, hosts, and platforms has been evaluated yet. This systematic review and meta-analysis aimed to evaluate the efficacy of vaccines against Flavobacterium infections in fish. We systematically searched PubMed, Web of Science, and Scopus for controlled challenge trials. The pooled risk ratio was calculated using a random effects model. Heterogeneity was assessed, and publication bias was evaluated by funnel plot and Egger's test. Sixty studies were included in this study, 59 of which were included in the main meta-analysis (377 comparisons) after screening. The overall pooled risk ratio was 0.572 (95% confidence interval: 0.55-0.60), corresponding to a relative percent survival of 42.8%, with high heterogeneity. Efficacy differed significantly by pathogen species, fish host, vaccine type and administration route. The highest protection by pathogen species was against F. davisii in channel catfish (relative percent survival = 86.8%), and the lowest against F. columnare in grass carp (relative percent survival = 30.6%). Live attenuated vaccines induced the highest protection, followed by inactivated and subunit vaccines. Among administration routes, immersion and oral vaccination achieved comparable protection, both outperforming injection. Significant publication bias was detected (P<0.001). Vaccination effectively reduces fish mortality from Flavobacterium infections, but its efficacy is highly context dependent. Taken together, standardized challenge protocols and improved reporting are needed. Priority should be given to developing effective vaccines against F. oreochromis and F. columnare.

microbiology↗

bioRxiv2026

Multi-hit STAG2 mutations define a high-risk subset of MDS and reveal convergent evolutionary targeting of cohesin

STAG2 is the most frequently mutated cohesin gene in myeloid neoplasms, yet the significance of multiple mutations within this X-linked tumor suppressor remains unknown. We analyzed a cohort of 1,967 adult patients with myeloid neoplasms and identified 233 cases (12%) harboring STAG2 mutations, including 38 cases (16%) with multiple STAG2 hits. Patients with multi-hit STAG2 mutations exhibited increased multilineage dysplasia compared with single-hit cases and experienced inferior overall survival, an effect driven primarily by patients with myelodysplastic syndromes (MDS). To investigate the molecular basis of recurrent STAG2 acquisition, we performed long-read sequencing in representative cases with phaseable STAG2 mutations. In the informative case examined, distinct truncating STAG2 mutations did not co-occur on the same DNA molecule, supporting independent acquisition rather than stepwise allelic inactivation. Cohort-level variant allele frequency patterns were consistent with recurrent evolutionary targeting of STAG2 across related clonal populations. Together, these findings support a model in which multi-hit STAG2 mutations arise through convergent evolution and define a biologically distinct, adverse-risk subset of MDS.

cancer biology↗

bioRxiv2026

DPCGS: a computational framework for linking GWAS to single-cell transcriptomics in complex traits and diseases

Complex traits and diseases arise from the interplay between genetic variation and cellular heterogeneity, making it essential to understand how genetic risk manifests at the cellular level. However, connecting genome-wide association studies (GWAS) to specific cell populations remains challenging due to cellular complexity and the prevalence of noncoding variants. Here, we present DPCGS, a computational framework that systematically integrates GWAS summary statistics with single-cell RNA-sequencing (scRNA-seq) data to identify trait-associated cell subpopulations, genes, and regulatory programs. Unlike existing approaches that primarily evaluate pathway enrichment or cell-type-level associations, DPCGS quantifies the enrichment of genetically prioritized genes within individual cells through a statistically calibrated gene-set scoring strategy, enabling high-resolution mapping of genetic risk to cellular states. Benchmarking across simulated and diverse human single-cell datasets demonstrates that DPCGS achieves superior accuracy, sensitivity, and robustness compared with existing methods, including scDRS and scPagwas. Applying DPCGS to Alzheimers disease and asthma reveals disease-associated cellular populations and uncovers potential molecular drivers, including CD74, FOS, and AP-1 family regulatory programs, providing insights into disease-specific immune and cellular mechanisms. By bridging genetic discoveries from GWAS with functional interpretation at single-cell resolution, DPCGS establishes a generalizable framework for dissecting the cellular architecture of complex traits and diseases. This approach enables systematic discovery of disease-relevant cell subpopulations, regulatory networks, and potential therapeutic targets, offering broad applications in human genetics, single-cell biology, and precision medicine.

bioinformatics↗

bioRxiv2026

A gap-free telomere-to-telomere genome of the purple-pericarp rice landrace Mojiang ZN65 resolves transposon-driven structural variation at the pericarp-pigmentation loci

Background: Black and purple pericarp in rice requires activation of the basic helix-loop-helix (bHLH) regulator Kala4/OsB2, and the known black-rice allele is a complex promoter rearrangement. Such repeat-embedded alleles can be difficult to resolve in fragmented assemblies, and for many pigmented landraces it remains unclear whether they carry that allele or structurally different ones. We asked three questions about the purple-pericarp landrace Mojiang ZN65: what is the structure of its Kala4/OsB2 upstream region; how frequent is gain of its upstream block in a rice diversity panel, and do the accessions with that gain share its junction; and how is it distributed relative to a second regulatory variant, a duplicated repeat in the OsKala3 promoter. Results: We assembled a gap-free telomere-to-telomere genome of ZN65 (395.1 Mb; all 24 telomeres; consensus quality value 53.6; 99.6% complete Benchmarking Universal Single-Copy Orthologues; indica), in which the flavonoid pathway showed no pathway-wide copy-number expansion. Upstream of Kala4/OsB2, a ~5.87-kb segment (block A) is present in three non-tandem copies, spanned as single molecules by three ultra-long Nanopore reads (115,450 bp); each of 33 other assemblies yielded a single homologous hit (10 of them coverage- or divergence-limited). This arrangement differs from the canonical black-rice Kala4 allele. Among 533 accessions of the 3,000 Rice Genomes panel, read depth detected block A gain in four accessions (three at three or more copies, 0.56%; one at two). All four carry the ZN65 junction at the same base, and three carry a pigment term in their variety names from a lexicon fixed in advance (Fisher's exact test, P = 0.0023). All four also have OsKala3 promoter-repeat read depth consistent with the two-unit configuration reported for black-pericarp cultivars, a state found in 1.9% of the panel. Conclusions: ZN65 carries a rare Kala4/OsB2 structural variant whose junction is shared with other accessions named as pigmented rice and which, in this panel, occurs only in accessions whose OsKala3 promoter-repeat read depth indicates duplication. Its effect on pigmentation remains to be tested with measured pericarp phenotypes and functional assays.

plant biology↗

bioRxiv2026

Research on Intelligent Optimization of Farm Planting Strategies Driven by Crop Simulation Models: A Case Study of Farm X

To meet the growing demand for precision and intelligent agricultural management, crop simulation models offer substantial potential for optimizing farm planting strategies. By simulating crop growth processes and assessing the effects of different management practices, these models provide a scientific basis for planting decision-making. In this study, the DSSAT model was first used to optimize the planting strategies of Farm X in 2023. Based on the optimized plans, the model was further applied to predict crop yields per unit area for 2024 and to establish the relationships among yield, planting density, and fertilizer application rate. Subsequently, SPSS was employed to develop a regression model describing the relationship among net profit per unit area, planting density, and fertilizer application rate. A genetic algorithm was then used to identify the optimal solutions under different scenarios, generating prescription maps for the optimal planting density and fertilizer application rate for each plot of Farm X in 2024. The results provide a scientific reference for the mechanized and automated implementation of field management practices and support the dual optimization of economic returns and resource use efficiency. This study not only conducted a systematic optimization of Farm X planting strategies for 2023, but also provided detailed predictions and optimized prescriptions for 2024 in a visual and practical form. The proposed approach offers a scientific decision-support tool for farm planting strategy formulation and lays a foundation for the intelligent and automated development of modern agriculture.

plant biology↗

bioRxiv2026

Dual-Engineered Dendritic Cell Derived Small Extracellular Vesicles Couple T Cell Priming with Checkpoint Reprogramming for Synergistic Immunotherapy

Immunotherapy has transformed cancer treatment, yet cell-based therapies remain complex and costly, and immune checkpoint blockade (ICB) agents often suffer from limited stability and poor T-cell selectivity. Here, we develop an engineered dendritic cell-derived small extracellular vesicle (DC-sEV) nanoplatform for combinatorial immunotherapy via in situ T-cell activation and checkpoint reprogramming. DC-sEVs preserve intrinsic dendritic-cell immunobiology, enabling antigen presentation and potent T-cell activation. We further integrate high-efficiency cargo loading and membrane functionalization to selectively deliver ICB payloads to T cells, achieving dual reprogramming that sustains effector function and amplifies antitumor immunity. This approach reduced cancer cell viability to 44.05% in vitro and produced 82.12% tumor growth inhibition in vivo, establishing DC-sEVs as a targeted, scalable cell-free immunotherapy platform. HIGHLIGHTSO_LIDC-sEVs preserve antigen presentation, T-cell activation, and lymph node targeting C_LIO_LIChirality-assisted loading with pH-responsive functionalization enables efficient cytosolic delivery while maintaining membrane bioactivity C_LIO_LIEngineered DC-sEVs combine in situ T-cell priming and PD-1 silencing to enhance effector function C_LIO_LIIn situ T-cell reprogramming drives potent antitumor efficacy and favorable tumor microenvironment remodeling C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/717283v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@e3b05org.highwire.dtl.DTLVardef@44febcorg.highwire.dtl.DTLVardef@1b00479org.highwire.dtl.DTLVardef@f5db2a_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2026

MTB-KB: A Curated Knowledgebase of Mycobacterium tuberculosis Related Studies

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), has regained its position as the worlds leading killer among infectious diseases. Despite extensive research progress across epidemiology, diagnosis, drug development, treatment regimens, vaccines, drug resistance, virulence factors, and immune mechanisms, MTB-related knowledge remains fragmented across thousands of publications, limiting its effective use. To address this gap, we present MTB-KB, a literature-curated knowledgebase that systematically integrates high-impact findings from eight major sections of TB research. The current release contains 75,170 associations from 1,246 publications, covering 18,439 entities standardized using authoritative databases and WHO-endorsed classifications. A central feature is the interactive knowledge graph, which links cross-section associations to reveal and infer MTB-host interactions, treatment strategies, and vaccine development opportunities. MTB-KB also provides a user-friendly interface with browsing, advanced search, and statistical visualization. Overall, by consolidating dispersed MTB knowledge into a structured and accessible platform, MTB-KB provides a valuable resource for researchers, clinicians, and policymakers, supporting both basic and clinical TB research, enabling evidence-based TB prevention, diagnosis, and treatment, and contributing to global elimination efforts. MTB-KB is accessible at https://ngdc.cncb.ac.cn/mtbkb/.

bioinformatics↗

bioRxiv2026

Spike-in probe-enhanced single-cell RNA-seq reveals post-infusion transcriptomic remodeling of "prime-and-kill" synNotch-CAR-T cells

We previously developed synthetic Notch (synNotch)-chimeric antigen receptor (CAR)-T cells to improve the safety and efficacy of CAR-T therapy for glioblastoma. In this system, an anti-EphA2/IL13R2-dual-CAR is expressed only upon recognition of tumor- or brain-specific "priming" antigens, EGFRvIII (termed E-SYNC cells) or brevican (B-SYNC), respectively, with E-SYNC currently under phase I clinical evaluation (NCT06186401). However, tracking and profiling these engineered cells in vivo remain challenging, limiting our understanding of their activity and therapeutic potential. To address this gap, we developed a single-cell RNA-sequencing (scRNA-seq) workflow with custom spike-in probes for synNotch-CAR transcripts, enabling simultaneous detection of engineered cells and transcriptomic profiling. In vitro, integration of multiple probes using machine-learning-assisted classifiers detected 78.2% of E-SYNC cells and 60.0% of B-SYNC cells with 98.0% specificity. In a xenograft model, synNotch-positive cells were detected across the spleen, lung, and brain, with the highest frequency and most robust priming and activation observed in the brain. Single-cell transcriptomic analyses revealed tissue-specific differentiation programs, including cytotoxicity, proliferation, metabolic activity, and acquisition of tissue-resident memory phenotypes, shaped by both environmental cues and synNotch-mediated antigen recognition. In summary, this spike-in probe-enhanced scRNA-seq workflow enables robust detection and high-resolution characterization of synNotch-CAR-T cell dynamics and provides a broadly applicable platform for monitoring engineered immune cells in diverse clinical contexts. One Sentence SummaryOur spike-in probe-enhanced single-cell RNA-sequencing method enables analysis of tissue-dependent activation and transcriptional states of synNotch-CAR-T cells, providing a robust and scalable platform for in vivo tracking and transcriptomic profiling of engineered cell therapies.

cancer biology↗

bioRxiv2026

Structure-guided discovery and engineering of miniature CRISPR-Cas12m for epigenome editing

CRISPR-based epigenome editing represents a programmable strategy to precisely modulate gene expression, holding immense promise for therapeutic applications. However, the large size of the dCas proteins substantially impedes the delivery via adeno-associated virus (AAV) vectors. Here, through iterative bioinformatics analysis, structure-guided predictions, and functional assays, we identified and characterized PmCas12m, a novel miniature subtype V-M CRISPR-Cas12m. PmCas12m exhibited flexible 5-YTN-3 PAM-dependent recognition and robust double-stranded DNA binding properties, while lacking DNA cleavage activity, thus positioning it as an ideal tool for epigenome editing. Cryogenic electron microscopy (cryo-EM) structures of PmCas12m unveiled its unique molecular mechanism of DNA binding facilitating interference. Guided by these structural insights, we employed deep mutational scanning (DMS) and protein engineering to develop xCas12m, a hypercompact variant with highly potent and specific epigenome editing capabilities in human cells. We further constructed the xCas12m-CRISPRoff platform in a single AAV vector, which achieved durable epigenetic silencing and effective inhibition of hepatitis B virus (HBV) infection in a mouse model. Collectively, these findings establish xCas12m as a versatile epigenome editing platform with transformative potential for treating diseases, paving the way for clinical translation of epigenetic therapies.

bioengineering↗

bioRxiv2026

Systematic identification of seed-driven off-target effects in Perturb-seq experiments

Genome-wide Perturb-seq (GWPS) has emerged as a powerful approach for unbiased mapping of gene regulatory networks. A key assumption underlying many Perturb-seq analyses is that each guide RNA exclusively perturbs a single target locus. Without methods to identify and filter off-target events, erroneous gene-pathway associations driven by off-target activity can propagate into downstream analyses. Here, we present a workflow for the systematic identification of candidate off-target events in CRISPRi Perturb-seq experiments. Our approach exploits the observation that cells harboring a guide which represses an off-target gene display transcriptional similarity to cells in which that gene is directly targeted by an on-target guide. We apply our workflow to multiple GWPS datasets and nominate off-target events in which a guide nominally targeting one gene also represses a distinct gene producing a phenotype likely attributable to the off-target perturbation. We use both off-target gene repression and guide seed sequence alignments at the off-target promoter locus as evidence for off-target effects and find independent evidence of putative off-target events in separate GWPS datasets. Together, these results establish a principled framework for the identification and filtering of off-target guide effects in Perturb-seq experiments.

genomics↗

bioRxiv2026

AI-designed cyclic peptides enable controllable modulation of the CD28 immune checkpoint

Immune checkpoint therapies have transformed immunotherapy but remain dominated by biologic agents characterized by prolonged receptor occupancy and limited pharmacologic controllability. Synthetic modalities capable of targeting protein-protein interaction interfaces while enabling tunable immune regulation remain largely unexplored. Here, we report an AI-guided strategy for discovering cyclic peptide antagonists of the costimulatory receptor CD28. The lead peptide, CIP-3, binds the CD28 extracellular domain with nanomolar affinity and competitively disrupts CD28-ligand interactions. In primary human immune systems, CIP-3 suppresses CD28-dependent T-cell activation without intrinsic agonist activity and exhibits rapid pharmacologic reversibility, enabling exposure-dependent control of immune signaling. In a T-cell transfer model of chronic colitis, CIP-3 confers dose-dependent therapeutic efficacy and reduces systemic inflammatory cytokines. CIP-3 also suppresses cytokine production across independent healthy donors and patient-derived PBMCs from individuals with ulcerative colitis with efficacy comparable to a benchmark anti-CD28 biologic. Together, these findings establish AI-designed cyclic peptides as a controllable synthetic modality for immune checkpoint modulation.

pharmacology and toxicology↗