Biology subjects

Bezney, J.

Publications and source records attributed to Bezney, J..

9 recordsLinked to original sources

bioRxiv2026

PHAROS: turning single-cell perturbation models into target-directed drug-combination screens

Combination therapies are central to cancer treatment, but exhaustive screening is impractical. We introduce PHAROS, a framework that turns a pretrained single-cell perturbation model into a target-directed search engine for drug combinations. PHAROS predicts how a cell population changes under a drug, one drug at a time, then chains these predictions together to simulate drug combinations. It scores each simulated outcome against the desired target state and uses a search algorithm to find the most promising combinations, all without retraining the underlying model. Across two independent combinatorial perturbation datasets, PHAROS recovered exact or mechanism-matched two-drug responses in cell lines, both seen and unseen during model training. Its rankings were specific to the requested conversion and were not explained by single-drug effects, additive effects, or shared mechanism of action. In exploratory analyses of patient-derived metastatic HR+/HER2$-$ breast tumors and basal cell carcinoma (BCC), PHAROS prioritized FDA-approved regimens, distinguished combinations by their predicted tumor-versus-immune objective profiles, and nominated pathway-level hypotheses, while explicitly identifying both tumor cohorts as outside the model's supported distribution. PHAROS provides a modular route from pretrained virtual-cell models to inverse, single-cell combination-screening platforms.

bioinformatics↗

bioRxiv2025

Innate immune responses to Plasmodium falciparum disrupt the blood-brain barrier

Plasmodium falciparum accumulation at the blood-brain barrier (BBB) is a hallmark of cerebral malaria, a life-threatening complication. Conversely, the contribution of the immune response to vascular injury has long been debated. Here, we studied the role of innate immune cells as potential effectors of vascular damage using a human in vitro 3D-BBB model. Parasite-stimulated immune cells from malaria-naive donors increased adhesion to microvessels, at least partly through LFA-1. This caused barrier disruption and inflammatory activation of BBB cells. Secretion of TNF-, IFN-{gamma}, and granzyme B by monocytes, NK and {gamma}{delta} T cells correlated with vascular injury, and accumulation of immune cells was required for local barrier damage. Our computational analysis disentangled pathogenic mechanisms driven specifically by either P. falciparum parasite or immune cells, as well as shared pathways. These findings demonstrate how vascular-immune interactions may contribute to vascular injury in cerebral malaria and point towards the potential of immunomodulatory therapeutics.

immunology↗

bioRxiv2025

Perturbing RNA localization for functional study in neurons

Spatial RNA organization plays a pivotal role in diverse cellular processes and diseases, but the functional implications of spatial RNA localization remain underexplored. We present CRISPR-mediated transcriptome organization (CRISPR-TO) that harnesses RNA-guided, nuclease-dead dCas13 for programmable control of RNA localization in live cells. CRISPR-TO enables targeted localization of RNAs to diverse subcellular compartments, including p-bodies, stress granules, telomeres, and nuclear stress bodies, across cell types. In primary cortical neurons, we demonstrate that repositioned mRNAs undergo local translation along neurites and at neurite tips and co-transport with ribosomes, with {beta}-actin mRNA localization enhancing the formation of dynamic filopodial protrusions and inhibiting axonal regeneration. Furthermore, CRISPR-TO-enabled parallel screening in primary neurons identifies Stmn2 mRNA localization as a driver of neurite outgrowth. By enabling large-scale perturbation of the spatial transcriptome, CRISPR-TO bridges a critical gap left by current sequencing and imaging technologies, offering a versatile platform for high-throughput functional interrogation of RNA localization in living cells and organisms.

bioengineering↗

bioRxiv2023

Simultaneous CRISPR screening and spatial transcriptomics reveals intracellular, intercellular, and functional transcriptional circuits.

Pooled optical screens have enabled the study of cellular interactions, morphology, or dynamics at massive scale, but have not yet leveraged the power of highly-plexed single-cell resolved transcriptomic readouts to inform molecular pathways. Here, we present Perturb-FISH, which bridges these approaches by combining imaging spatial transcriptomics with parallel optical detection of in situ amplified guide RNAs. We show that Perturb-FISH recovers intracellular effects that are consistent with Perturb-seq results in a screen of lipopolysaccharide response in cultured monocytes, and uncover new intercellular and density-dependent regulation of the innate immune response. We further pair Perturb-FISH with a functional readout in a screen of autism spectrum disorder risk genes, showing common calcium activity phenotypes in induced pluripotent stem cell derived astrocytes and their associated genetic interactions and dysregulated molecular pathways. Perturb-FISH is thus a generally applicable method for studying the genetic and molecular associations of spatial and functional biology at single-cell resolution.

genomics↗

bioRxiv2023

Single-cell multi-omics map of human foetal blood in Down's Syndrome

Downs Syndrome (DS) predisposes individuals to haematological abnormalities, such as increased number of erythrocytes and leukaemia in a process that is initiated before birth and is not entirely understood. To understand dysregulated hematopoiesis in DS, we integrated single-cell transcriptomics of over 1.1 million cells with chromatin accessibility and spatial transcriptomics datasets using human foetal liver and bone marrow samples from three disomic and 15 trisomic foetuses. We found that differences in gene expression in DS were both cell type- and environment-dependent. Furthermore, we found multiple lines of evidence that DS haematopoietic stem cells (HSCs) are "primed" to differentiate. We subsequently established a DS-specific map of enhancer-gene relationships in disomic and trisomic HSCs using 10X Multiome data. By integrating this map with genetic variants associated with blood cell variation, we discovered that trisomy restructured enhancer-gene maps to dysregulate enhancer activity and gene expression critical to erythroid lineage differentiation. Further, as DS mutations display a signature of oxidative stress, we validated both increased mitochondrial mass and oxidative stress in DS, and observed that these mutations preferentially fell into regulatory regions of expressed genes in HSCs. Altogether, our single- cell, multi-omic resource provides a high-resolution molecular map of foetal haematopoiesis in Downs Syndrome and indicates significant enhancer-gene restructuring giving rise to co- occurring haematological conditions.

genomics↗

bioRxiv2023

Compressed Perturb-seq: highly efficient screens for regulatory circuits using random composite perturbations

Pooled CRISPR screens with single-cell RNA-seq readout (Perturb-seq) have emerged as a key technique in functional genomics, but are limited in scale by cost and combinatorial complexity. Here, we reimagine Perturb-seqs design through the lens of algorithms applied to random, low-dimensional observations. We present compressed Perturb-seq, which measures multiple random perturbations per cell or multiple cells per droplet and computationally decompresses these measurements by leveraging the sparse structure of regulatory circuits. Applied to 598 genes in the immune response to bacterial lipopolysaccharide, compressed Perturb-seq achieves the same accuracy as conventional Perturb-seq at 4 to 20-fold reduced cost, with greater power to learn genetic interactions. We identify known and novel regulators of immune responses and uncover evolutionarily constrained genes with downstream targets enriched for immune disease heritability, including many missed by existing GWAS or trans-eQTL studies. Our framework enables new scales of interrogation for a foundational method in functional genomics.

systems biology↗

bioRxiv2022

A CRISPR/Cas9-based enhancement of high-throughput single-cell transcriptomics

Single-cell transcriptomics suffers from lapses in coverage of the full transcriptome, providing an incomplete gene expression profile of the cell. Here, we introduce single-cell CRISPRclean (scCLEAN), an in vitro molecular method that can be universally inserted into any single-cell RNA-seq workflow to improve the sensitivity of the assay. Utilizing CRISPR/Cas9, scCLEAN works to selectively remove highly abundant uninformative molecules, redistributing ~50% of reads to enrich for lowly expressed transcripts. Utilizing immune cells, we describe a validation of scCLEAN showing a 2.1-fold enrichment in library complexity with negligible off-target effects. Subsequently, applying scCLEAN to single-cell iso-seq samples results in a 4.6-fold improvement in unique isoform detection. Thus, demonstrating a benefit in short and long read sequencing applications. Finally, we illustrate the ability of scCLEAN to elucidate biological insights by applying it to two participant cohorts of cardiovascular samples, bringing to light novel molecular characteristics including inflammatory signatures.

genetics↗

bioRxiv2020

Targeted DNA Damage Repair CRISPR/Cas9 Knockout Screen Identifies Novel Classification of Poly-ADP Ribose Polymerase Inhibitors Based on Key Base Excision Repair Proteins

DNA repair deficiencies have become an increasingly promising target for novel therapeutics within the realm of clinical oncology. Recently, several inhibitors of Poly(ADP-ribose) Polymerases (PARPs) have received approval for the treatment of cancers primarily with deleterious mutations in the homologous recombination (HR) proteins, BRCA1 and BRCA2. Despite numerous clinical trials which have been completed or are currently ongoing, the mechanism of action by which PARP inhibitors selectively kill tumor cells is poorly understood. While many believe "trapping" of PARP proteins to DNA at sites of damage is the most important determinant driving cytotoxicity by these drugs, clinically effective inhibitors exist with a diverse range of PARP-trapping qualities. These findings suggest that characterization of inhibitors as strong versus weak trappers does not properly capture the intra-class characteristics of these drugs. Here, we use a novel, targeted DNA damage response (DDR) CRISPR/Cas9 screening library to reveal heterogenous genetic dependencies on the base excision repair (BER) pathway for PARP inhibitors, which is not correlated with PARP trapping ability or catalytic inhibition of PARP. These findings demonstrate that inhibition of PARylation and induction of PARP trapping are not the only factors contributing to distinct biological activity for different PARP inhibitors, and they provide insight into the optimal choice of PARP inhibitors for use in the setting of specific DDR defects. AUTHOR SUMMARYTargeted cancer therapies rely on our general understanding of which genetic mutations are involved in both sensitivity and resistance to such anticancer agents. In this study, we describe the use of functional genetic screening to evaluate the role of various DNA repair proteins in response to inhibitors of PARP, a quintessential example of targeted therapy. While PARP inhibitors are best known for their utility in cancers with homologous recombination defects, we show that some inhibitors within this class may have additional functionality in cancers with deficient base excision repair. These findings highlight not only the importance of PARP inhibitor selection in the appropriate context, but also the mechanistic differences that exist within this class of inhibitors. It is our hope that our findings will inspire future work evaluating the use of specific PARP inhibitor selection in designing clinical trials to further expand the use of PARP inhibitors beyond tumors with homologous recombination deficiencies.

cancer biology↗

bioRxiv2019

Compressed sensing for imaging transcriptomics

Tissue and organ function rely on the organization of cells and molecules in specific spatial structures. In order to understand these structures and how they relate to tissue function in health and disease, we would ideally be able to rapidly profile gene expression over large tissue volumes. To this end, in recent years multiple molecular assays have been developed that can image from a dozen to ~100 individual proteins1-3 or RNAs4-10 in a sample at single-cell resolution, with barcodes to allow multiplexing across genes. These approaches have serious limitations with respect to (i) the number of genes that can be studied; and (ii) imaging time, due to the need for high-resolution to resolve individual signals. Here, we show that both challenges can be overcome by introducing an approach that leverages the biological fact that gene expression is often structured across both cells and tissue organization. We develop Composite In Situ Imaging (CISI), that combines this biological insight with algorithmic advances in compressed sensing to achieve greater efficiency. We demonstrate that CISI accurately recovers the spatial abundance of each of 37 individual genes from 11 composite measurements in 12 bisected mouse brain coronal sections covering 180mm2 and 476,276 cells without the need for spot-level resolution. CISI achieves the current scale of multiplexing with two orders of magnitude greater efficiency, and can be leveraged in combination with existing methods to multiplex far beyond current scales.

molecular biology↗