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CRISPR

CRISPR: explore 11 source-linked works published from 2026 to 2026, with original documents and citations.

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Sources: biorxiv. Collection updated 2026-09-15. Counts describe this index, not the complete source archives.

A patient-derived LMX1B variant causes tissue-specific manifestations of nail-patella syndrome in mice

Nail-patella syndrome (NPS) is a multisystem disorder caused by pathogenic variants in LMX1B and is characterized by dysplasia of the nails and patellae as well as extraskeletal complications such as progressive nephropathy and glaucoma. We generated a CRISPR/Cas9 knock-in mouse carrying the R252Q substitution, corresponding to a human LMX1B variant associated with renal-predominant disease. Phenotypic analysis revealed that homozygous mice were viable, but they displayed marked growth retardation and severe bilateral ocular opacity. Interestingly, while this model exhibited clear skeletal and ocular defects, the renal phenotype was relatively mild, although increased urinary albumin excretion, focal glomerular basement membrane abnormalities, and subtle changes in renal gene expression were detected. Beyond the classical NPS hallmarks, mutant mice also displayed midbrain morphological abnormalities, suggesting broader developmental consequences of this LMX1B variant. This patient-derived variant model not only recapitulates the pleiotropic features of NPS but also demonstrates organ-specific susceptibility to the R252Q substitution, providing a foundation for elucidating the complex molecular mechanisms underlying multisystem disease.

genetics

Phosphorylation of spleen tyrosine kinase Y130 positively regulates intracellular signaling and functional responses in platelets

Syk is a non-receptor type protein-tyrosine kinase (PTK), which is associated with platelets surface receptors, glycoprotein VI (GPVI) and C-type lectin-like receptor II-type (CLEC-2). Syk is also expressed in most hematopoietic lineage cells and other cells, such as fibroblasts and neuronal cells. Syk has two tandem SH2 motifs and a C-terminal kinase domain, which are interrupted by interdomains A and B containing multiple tyrosine residues playing a regulatory role upon phosphorylation. This study aims to evaluate the role of Y130 in Syk signaling in platelets. Syk(Y130F) knock-in (KI) mice we generated using the CRISPR-Cas9 technique represent the first in-vivo model harboring this mutation. Using this system, we compared the platelet signaling and responses in wild-type (WT) and Syk(Y130F) littermates. Platelets from homozygous Syk(Y130F) mice showed a decrease in functional responses after activation with CRP, a GPVI agonist, and CLEC-2 crosslinking compared to WT littermates with no significant differences in responses to PAR-4 or purinergic receptor agonists. Key signaling events triggered via both GPVI and CLEC-2, including phosphorylation LAT and PLC-2, were also reduced in Syk(Y130F) platelets at low agonist concentrations. Consistent with these findings, the time to occlusion in the FeCl3 injury model and bleeding time in the tail bleeding assay were significantly enhanced in Syk(Y130F) mice compared to WT littermates. Thus, phosphorylation of Syk Y130 enhances GPVI- and CLEC-2-mediated signaling and functional responses in platelets affecting thrombosis and hemostasis.

molecular biology

The role of mu opioid receptors on excitatory and inhibitory neurons in the rostral ventromedial medulla in neuropathic pain

Descending projections from the brain to the spinal cord can regulate painful stimulus processing and are modulated by endogenous and exogenous opioids. We investigated the role of mu opioid receptors (MORs) in GABAergic vs. glutamatergic neurons of the rostral ventral medulla (RVM) in a mouse model of chronic neuropathic pain. We found that activating glutamatergic and GABAergic neurons in the RVM both result in antinociception [BC1.1]at baseline, but glutamatergic neurons enhance pain responses after nerve injury. [BC2.1]We then interrogated the role of RVM MOR signaling on neuropathic pain by using CRISPR/Cas9 to delete MOR in glutamatergic or GABAergic RVM neurons. We found that MOR knockout in glutamatergic and GABAergic RVM neurons precipitates early neuropathic pain onset with no effect on chronic pain intensity. These results suggest that RVM MOR signaling modulates hypersensitivity in the early phase of injury, but chronic neuropathic pain is largely independent of mu opioid receptor signaling.

neuroscience

Antibody-dependent priming of spontaneous germinal centers by autoreactive B cells

Autoreactive germinal centers (GCs) are central to autoimmune pathogenesis, yet the mechanisms by which single autoreactive B cell clones prime systemic autoimmunity remain unclear. Using the 564Igi mixed chimera model, we demonstrate that autoreactive 564Igi B cells break tolerance in wild-type B cells through an unexpected mechanism independent of cognate T cell interactions. While B cell-intrinsic TLR7 signaling was essential for spontaneous GC formation, deletion of MHC class II, CD40, or CD80/86 on GC-priming 564Igi B cells failed to prevent GCs. Instead, CRISPR-mediated deletion of Prdm1 (encoding BLIMP-1) in 564Igi B cells ablated spontaneous GCs, implicating autoantibody production as the primary driver. These findings reveal that autoantibodies can initiate feed-forward mechanisms that propagate systemic autoimmunity, independent of B cell-intrinsic antigen presentation.

immunology

m1A58 acts as a conformational checkpoint coupling human initiator tRNA maturation to translation initiation

tRNAs are characterized by extensive chemical modifications that influence tRNA fate. N1-methyladenosine at position 58 (m1A58) is a widespread core tRNA modification linked to physiological and pathological processes. However, how m1A58 coordinate tRNA folding and processing to ensure translational efficiency in mammalian cells remains largely unknown. Using acute dTAG-mediated degradation and CRISPR-Cas9 knockout, we identified initiator methionine tRNA (tRNAiMet) as selectively vulnerable to m1A58 loss, lacking the isodecoder buffering observed for most other tRNA isoacceptors. NMR analysis of the tRNAiMet showed that m1A58 stabilizes D/T-loop interactions, consistent with a maturation-competent conformation. In vitro processing assays further demonstrated that m1A58 promotes RNase P-mediated 5'-leader removal and RNase Z-mediated 3'-trailer cleavage, while La/SSB protects accumulated precursors. Disrupting this checkpoint impaired the assembly of the eIF2-containing 43S pre-initiation complex and global protein synthesis, which was substantially rescued by adding m1A58-modified tRNAiMet. Acute TRMT6 degradation elicited temporally coordinated gene-expression responses involving proteostasis, transport and signaling. Together, these findings establish m1A58 as a conformational checkpoint coupling human initiator-tRNA maturation to translation initiation and stress responses.

molecular biology

Integrated Transcriptomic and CRISPR Dependency Analysis Prioritizes a CDK1-AURKB Mitotic Vulnerability Axis in Diffuse Intrinsic Pontine Glioma

Diffuse intrinsic pontine glioma (DIPG), now classified within diffuse midline glioma, H3K27-altered, remains a lethal pediatric brainstem tumor with limited therapeutic options. Here, we integrated public DIPG transcriptomic datasets, protein-protein interaction modeling, functional enrichment, immune deconvolution, survival analysis, and DepMap CRISPR dependency data to nominate candidate mitotic vulnerabilities. Differential expression analysis comparing 27 DIPG tumors with 6 brainstem low-grade glioma comparator samples identified a proliferative transcriptional program enriched for chromosome segregation, nuclear division, and cell-cycle pathways. Network analysis prioritized a compact mitotic hub module containing CDK1, AURKB, TOP2A, CDC20, CDCA8, and related G2/M regulators. CIBERSORT analysis of an independent DIPG cohort inferred low cytotoxic T-cell signal, consistent with an immune-cold phenotype, although immune-cell fractions require orthogonal validation. Survival analysis showed that neither inferred immune scores nor a composite mitotic hub score significantly stratified overall survival. DepMap CRISPR gene-effect data nominated CDK1, AURKB, TOP2A, and BIRC5 as candidate dependencies across brain tumor models. These findings provide a computational framework for prioritizing mitotic vulnerabilities in DIPG and support experimental validation in disease-relevant models.

bioinformatics

ENPP3 expressed by HER2-positive breast cancer cells is associated with good prognosis by restraining epithelial-to-mesenchymal phenotype

Background Ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3/CD203c) is largely studied as a marker of mast cells and basophils. By depleting extracellular ATP, it prevents excessive activation of mast cells and basophils, hence reducing inflammation and allergic reactions. Recent findings have also shown that Enpp3 can deplete cGAMP, another molecule involved in STING activation and IFN-mediated pro-inflammation. Little is still known regarding the role of Enpp3 in non-immune cells although a few reports have described its expression in healthy tissues and tumors. Methods In silico analysis were performed to investigate the expression levels and the prognostic value of Enpp3 in breast cancer, together with ovarian, prostate and colon carcinoma. ENPP3 expression was evaluated in formalin-fixed, paraffin-embedded tumor samples of breast cancer patients by immunohistochemistry, and in mouse mammary cancer cell lines by western blots. Cells were treated with EGFR ligands to stimulate the EGFR/HER2 axis. A mouse-derived mammary cancer cell line was engineered by CRISPR/Cas9 to introduce a GFP sequence under the control of the Enpp3 promoter. GFP-positive and -negative cells were sorted and analyzed by gene expression profiling to identify genes and pathways associated with Enpp3 expression. Finally, wild type and Enpp3 knockout cells were injected in the fat pad of Wsh mice, which do not have mast cells, to evaluate the growth of the tumors which were further analyzed by immunohistochemistry. Results We provide evidence that HER2-positive cells express higher levels of ENPP3 in samples of breast cancer patients. Moreover, in vitro models confirmed that HER2 expression and EGFR stimulation result in up-regulation of Enpp3. We identified pathways that can concur to Enpp3 expression and showed that in vivo the absence of Enpp3 promotes tumor growth and development of tumors with a marked epithelial-to-mesenchymal phenotype. Finally, in a small cohort of HER2-positive breast cancer patients, we found that ENPP3 expression correlates with increased relapse-free survival. Conclusions Despite its potential immunosuppressive role, our findings support the notion that ENPP3 expression is promoted by HER2 in breast cancer, and that it is endowed with a positive prognostic value.

cancer biology

Cas12a cleavage and trimming kinetics reveal mismatches as a tool to steer editing

Gene knockouts by CRISPR-Cas nucleases rely on targeted DNA cleavage and error-prone DNA repair: end-joining pathways can introduce insertions and deletions that assist in disrupting the coding sequence. However, only a fraction of edits achieves this, and an unfavorable array of repair outcomes typically requires switching to another editing technology. Key factors that influence repair are the types and lengths of DNA ends following cleavage. Here, we investigated Cas12a's ability to produce different ends and if they can be used to redistribute editing outcomes. We determined the sites and rates of target cleavage by Cas12a in vitro by combining kinetic modeling with nucleotide-resolution assays. For the first time, we show that trimming - repeated cleavage of an already cut target - occurs about 4x faster than initial cleavage; it also presents alternative DNA end structures for cellular repair. We next introduced specific mismatches to the gRNA. Cas12a maintained fast target cleavage, but changed where the target was cleaved and how quickly it was trimmed, compared to matched gRNA. We exploited the differences in cleavage dynamics between matched and mismatched gRNAs to develop reprogrammed gRNAs, i.e. rpgRNAs. Intentionally-mismatched rpgRNAs retained the high editing efficiency observed with traditional gRNAs. However, they redirected editing between in-frame and out-of-frame outcomes to enhance gene knockout success across genes. Reprogrammed gRNAs offer an efficient way to steer editing toward such preferred outcomes, while retaining the simplicity of gene editing with CRISPR-Cas nucleases.

biochemistry

HIF1A recruits primate-specific endogenous retroviruses into the human hypoxic and immune responses

Oxygen availability varies profoundly across the human body and changes further during inflammation, infection, tissue injury and disease. Immune cells must therefore continuously adapt their transcriptional and metabolic state based on the oxygen availability to them. Hypoxia-inducible factor 1 (HIF1A) is central to this adaptation and a marker of the cellular response to low oxygen, yet its genomic targets have been assembled from a non-repetitive fraction of the genome, leaving nearly half of the human genome largely unexplored. Here we define the gene and transposable-element (TE) landscape of the human hypoxic response across different human tissues, cell lines, and conditions. This directional TE response was reproduced in transformed cells and in primary immune cells isolated from blood and the physiologically oxygen-restricted tonsil. Single-cell profiling of peripheral blood mononuclear cells (PBMC) under hypoxia, pharmacological HIF stabilization, and interferon stimulation revealed a striking difference between the gene and retrotranscriptome responses. While gene responses were strongly cell-type dependent and in a bidirectional manner, TEs were overwhelmingly activated. This pattern extended to blood and tonsil immune cells, where ~70-90% of tested TE families were induced under hypoxia, with activated tonsil cells showing exclusively induced significant families, including THE1B, alongside increased LTR7 and HERVH. Integrating HIF1A ChIP-seq with transcriptional responses revealed that HIF1A does not engage repetitive DNA indiscriminately. Instead, its binding converged on LTR7, the promoter long terminal repeat of the HERVH endogenous retrovirus. Approximately 80% of HIF1A-bound LTR7 elements contained a canonical hypoxia-response element, and disruption of HIF1A DNA binding dramatically reduced the expression of occupied HERVH loci. CRISPR deletion of individual LTR7/HERVH loci altered the expression of distant and neighboring genes, demonstrating that hypoxia-responsive retroelements can participate directly in host gene regulation and contribute to overall physiology. Our findings reveal the repetitive genome as a previously underappreciated component of oxygen sensing. We propose that HIF1A recruits selected endogenous retroviral elements into the human hypoxic response, extending oxygen-dependent regulation beyond conventional gene promoters and providing an additional regulatory layer through which tissue oxygenation can shape immune-cell state and human physiology.

genomics

BARCS: beta-binomial regression for multivariate CRISPR screen design

Pooled CRISPR screens increasingly use longitudinal, donor-adjusted, and factorial designs, but beta-binomial screen methods have largely remained limited to pairwise comparisons. BARCS extends the library-total-conditional beta-binomial model to guide-level regression with an arbitrary design matrix, enabling direct estimation of time, covariate, and interaction effects. In four replicate-complete Cas13 screens, adding the intermediate time point modestly improved essential-gene recovery. Applying the same non-targeting-control scaling rule to BARCS, MAGeCK-MLE, edgeR-QL, DESeq2, and limma--voom produced similar calibration across all five methods, while the four alternatives ranked essential genes more strongly than BARCS. In an ordered-bin IL2RA screen, donor-adjusted BARCS recovered more validated regulators with fewer total calls than the matched four-bin MAGeCK-MLE fit, and cross-fitted controls exposed excess guide-level significance. Simulations showed gains from dispersion moderation and control-based denominators, but seed-specific results exposed denominator sensitivity and a null grid localized substantial gene-level error to correlated-guide aggregation rather than dispersion alone. Aggregation-matched control scaling reduced but did not eliminate this error. An external audit prompted by concerns about beta-binomial false discoveries showed that the reported CB2 null-discovery count disappeared when full-library totals were restored. This corrected one denominator-dependent result but did not refute the broader calibration concern; nominal-level calibration remained unresolved. BARCS therefore contributes a multivariable extension of the library-total-conditional beta-binomial model together with an explicit account of where its inference is valid: guide-level coefficients are supported by independent biological libraries, whereas gene-level summaries and partitioned-bin designs require correlation-aware aggregation or joint modelling that the present implementation provides diagnostically rather than generatively. We report this boundary because complex pooled designs make it consequential, not because it is unique to the beta-binomial model.

bioinformatics

Model-based evaluation of Targeted-Antibacterial-Plasmids (TAPs) transfer kinetics and resensitization of pOXA-48 carbapenem-resistant Escherichia coli

Background Targeted-Antibacterial Plasmids (TAPs) are engineered mobile genetic elements that use bacterial conjugation to deliver selective CRISPR/Cas9 antibacterial activity against a specific target strain. Yet, the efficiency of TAPs is typically evaluated at a single time point, whereas the success of TAP-mediated resensitization critically depends on the dynamics of plasmid transfer and the complex interactions between bacterial subpopulations. This is the first study to evaluate the efficiency of a conjugation-based antibacterial approach at the subpopulation level, using an analytical framework analogous to that used for conventional antibiotics. Here, we investigate which process limits resensitization by TAPF-dCas9-OXA48: plasmid delivery, dCas9 activity, or the emergence of refractory and escape populations. Methods We fitted a mechanistic model of five interacting subpopulations (donors, recipients, transconjugants, escapers, and recusants) to 44 longitudinal conjugation experiments and used the fitted model to explore a range of biologically relevant scenarios. Results Using longitudinal conjugation data spanning 24 h, we show that up to 24% of recipients become recusants within 24h, refractory to further conjugation via entry exclusion, while secondary transconjugant emergence stays below 0.01%. Overall resensitization efficiency reaches up to 80%. Conclusion Plasmid transfer, rather than dCas9 repression, therefore appears to be the main bottleneck limiting the efficiency of TAPF-dCas9-OXA48 efficiency. These results identify plasmid delivery as a key engineering target for improving the performance of future TAPs.

bioinformatics
Compare source metadata on this page
WorkPublishedSource identifierSource
A patient-derived LMX1B variant causes tissue-specific manifestations of nail-patella syndrome in mice2026-09-0210.64898/2026.08.28.747744v1biorxiv
Phosphorylation of spleen tyrosine kinase Y130 positively regulates intracellular signaling and functional responses in platelets2026-09-0210.64898/2026.08.31.748427v1biorxiv
The role of mu opioid receptors on excitatory and inhibitory neurons in the rostral ventromedial medulla in neuropathic pain2026-09-0110.64898/2026.08.26.747135v1biorxiv
Antibody-dependent priming of spontaneous germinal centers by autoreactive B cells2026-09-0110.64898/2026.08.27.745373v1biorxiv
m1A58 acts as a conformational checkpoint coupling human initiator tRNA maturation to translation initiation2026-09-0110.64898/2026.08.28.747798v1biorxiv
Integrated Transcriptomic and CRISPR Dependency Analysis Prioritizes a CDK1-AURKB Mitotic Vulnerability Axis in Diffuse Intrinsic Pontine Glioma2026-09-0110.64898/2026.08.30.748149v1biorxiv
ENPP3 expressed by HER2-positive breast cancer cells is associated with good prognosis by restraining epithelial-to-mesenchymal phenotype2026-09-0110.64898/2026.08.31.747828v1biorxiv
Cas12a cleavage and trimming kinetics reveal mismatches as a tool to steer editing2026-09-0110.64898/2026.08.31.748204v1biorxiv
HIF1A recruits primate-specific endogenous retroviruses into the human hypoxic and immune responses2026-09-0110.64898/2026.08.31.748411v1biorxiv
BARCS: beta-binomial regression for multivariate CRISPR screen design2026-09-0110.64898/2026.08.31.748412v1biorxiv
Model-based evaluation of Targeted-Antibacterial-Plasmids (TAPs) transfer kinetics and resensitization of pOXA-48 carbapenem-resistant Escherichia coli2026-09-0110.64898/2026.09.01.748484v1biorxiv

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