Biology subjects

Osborne, T.

Publications and source records attributed to Osborne, T..

3 recordsLinked to original sources

bioRxiv2025

UNCOVERseq Enables Sensitive and Controlled Gene Editing Off-Target Nomination Across CRISPR-Cas Modalities and Systems

The rapid development of CRISPR-Cas gene editing technologies has revolutionized genetic medicine, offering unprecedented precision and potential for treating a wide array of genetic disorders. However, assessing the risks of unintended gene editing effects remains critical, and is complicated by new editing modalities and unclear analytical guidelines. We present UNCOVERseq (Unbiased Nomination of CRISPR Off-target Variants using Enhanced RhPCR), an improved in cellulo off-target nomination workflow designed to sensitively nominate off-target sites (<0.01% editing) with defined input requirements and analytical process controls to provide empirical performance evidence across diverse circumstances. Using this workflow, we nominated off-targets across 192 guide RNAs (gRNAs) and demonstrated superior performance compared to existing methodologies. We identified a subset of six gRNAs with a dynamic range of specificity and confirmed the relevance and high true positive rate of our nomination method, providing relative risk assessments for multiple modalities (S.p. Cas9 and derived high-fidelity variants / base editors) in a translational system involving hematopoietic stem and progenitor cells (HSPCs). Additionally, we established that double-strand break (DSB) editing retains a strong, positive rank correlation to single-strand break (SSB)-mediated base editing, highlighting the importance of DSB nomination sites as candidate loci for base editing. Overall, UNCOVERseq improves informed risk assessment of gene editing in translational systems by enhancing the quality of off-target nomination.

molecular biology↗

bioRxiv2025

Setdb2 Regulates Inflammatory Trigger-Induced Trained Immunity of Macrophages Through Two Different Epigenetic Mechanisms

"Trained immunity" of innate immune cells occurs through a sequential two-step process where an initial pathogenic or sterile inflammatory trigger is followed by an amplified response to a later un-related secondary pathogen challenge. The memory effect is mediated at least in part through epigenetic modifications of the chromatin landscape. Here, we investigated the role of the epigenetic modifier Setdb2 in microbial ({beta}-glucan) or sterile trigger (Western-diet-WD/oxidized-LDL-oxLDL)-induced trained immunity of macrophages. Using genetic mouse models and genomic analysis, we uncovered a critical role of Setdb2 in regulating proinflammatory and metabolic pathway reprogramming. We further show that Setdb2 regulates trained immunity through two different complementary mechanisms: one where it positively regulates glycolytic and inflammatory pathway genes via enhancer-promoter looping, and is independent of its enzymatic activity; while the second mechanism is associated with both increased promoter associated H3K9 methylation and repression of interferon response pathway genes. Interestingly, while both mechanisms occur in response to pathogenic training, only the chromatin-looping mechanism operates in response to the sterile inflammatory stimulus. These results reveal a previously unknown bifurcation in the downstream pathways that distinguishes between pathogenic and sterile inflammatory signaling responses associated with the innate immune memory response and may provide potential therapeutic opportunities to target cytokine vs. interferon pathways to limit complications of chronic inflammation.

immunology↗

bioRxiv2023

Targeting sterol regulatory element binding proteins (SREBPs) activation lowers intraocular pressure by halting its mechanosensing function in the trabecular meshwork

Trabecular meshwork (TM) cells are highly contractile and mechanosensitive to aid in maintaining intraocular pressure (IOP) homeostasis. Lipids are attributed to modulating TM contractility with poor mechanistic understanding. In this study using human TM cells, we identify the mechanosensing role of the transcription factors sterol regulatory element binding proteins (SREBPs) involved in lipogenesis. By constitutively activating SREBPs and pharmacologically inactivating SREBPs, we have mechanistically deciphered the attributes of SREBPs in regulating the contractile properties of TM. The pharmacological inhibition of SREBPs by fatostatin and molecular inactivation of SREBPs ex vivo and in vivo respectively results in significant IOP lowering. As a proof of concept, fatostatin significantly decreased the SREBPs responsive genes and enzymes involved in lipogenic pathways as well as the levels of the phospholipid, cholesterol, and triglyceride. Further, we show that fatostatin mitigated actin polymerization machinery and stabilization, and decreased ECM synthesis and secretion. We thus postulate that lowering lipogenesis in the TM outflow pathway can hold the key to lowering IOP by modifying the TM biomechanics. SynopsisIn this study, we show the role of lipogenic transcription factors sterol regulatory element binding proteins (SREBPs) in the regulation of intraocular pressure (IOP). (Synopsis Figure - Created using Biorender.com) O_LISREBPs are involved in the sensing of changes in mechanical stress on the trabecular meshwork (TM). SREBPs aid in transducing the mechanical signals to induce actin polymerization and filopodia/lamellipodia formation. C_LIO_LISREBPs inactivation lowered genes and enzymes involved in lipogenesis and modified lipid levels in TM. C_LIO_LISREBPs activity is a critical regulator of ECM engagement to the matrix sites. C_LIO_LIInactivation of SCAP-SREBP pathway lowered IOP via actin relaxation and decreasing ECM production and deposition in TM outflow pathway signifying a novel relationship between SREBP activation status and achieving IOP homeostasis. C_LI

cell biology↗