Biology subjects

Hwang, B.

Publications and source records attributed to Hwang, B..

7 recordsLinked to original sources

bioRxiv2026

A genome-wide CRISPR activation map of surface protein expression in human CD4 T cells

Surface proteins define T cell identity and function, but the abundance of each protein is not determined by transcription alone. Existing genome-wide CRISPR screens in primary human T cells either profile the transcriptome or isolate cells based on a single functional or protein phenotype. Here we present SCITO-Perturb-seq, a novel platform that couples combinatorial-indexed single-cell cytometry sequencing with pooled CRISPR activation (CRISPRa) to map the causal regulation of 201 surface proteins across 3.6 million human CD4 T cells. We find that 16% of activated genes significantly alter the expression of at least one surface protein. By applying semi-nonnegative matrix factorization to the perturbation effect matrix, we identified five modules corresponding to known CD4 T cell states. Notably, these modules group surface proteins by their shared response to perturbation, revealing coordinated regulation of proteins that are not co-expressed in unperturbed cells. SCITO-Perturb-seq represents the first genome-wide CRISPRa screen paired with direct, high-dimensional surface protein profiling, providing a comprehensive regulatory map of the CD4 T cell surface proteome.

genomics↗

bioRxiv2025

Mitochondrial Dysfunction Drives Age-Related Degeneration of the Thoracic Aorta

This study investigated the role of mitochondrial function in aortic aging. As the aorta ages, it becomes stiffer and less compliant, increasing the risk of aneurysmal disease, hypertension, and diastolic dysfunction. Given the role of mitochondrial dysfunction in non-age related aortopathies and as a hallmark of aging, we investigated its contribution to the aging aorta. Both male and female young (5-6 month) and aged (24-25 month) C57Bl/6J mice received mitochondrial-targeted peptide elamipretide (ELAM; SS-31) for 8 weeks. ELAM restored complex II-linked respiration in aged mice to values seen in young mice, while also improving relative phosphorylative flux. ELAM treatment also reduced inflammatory MMP9 expression and elastin breaks in aged mice. Bulk RNAseq analysis revealed that ELAM treatment significantly affected the aortic transcriptome in an age-dependent manner, reducing the expression of senescent and associated pro-inflammatory genes. Mitochondrial dysfunction thus drives aortic aging and is a potential therapeutic target for future study. TeaserLoss of mitochondrial function with age drives degeneration of the aging aorta, and thus is a potential target for therapy.

cell biology↗

bioRxiv2023

Mitigation of chromosome loss in clinical CRISPR-Cas9-engineered T cells

CRISPR-Cas9 genome editing has enabled advanced T cell therapies, but occasional loss of the targeted chromosome remains a safety concern. To investigate whether Cas9-induced chromosome loss is a universal phenomenon and evaluate its clinical significance, we conducted a systematic analysis in primary human T cells. Arrayed and pooled CRISPR screens revealed that chromosome loss was generalizable across the genome and resulted in partial and entire loss of the chromosome, including in pre-clinical chimeric antigen receptor T cells. T cells with chromosome loss persisted for weeks in culture, implying the potential to interfere with clinical use. A modified cell manufacturing process, employed in our first-in-human clinical trial of Cas9-engineered T cells,1 dramatically reduced chromosome loss while largely preserving genome editing efficacy. Expression of p53 correlated with protection from chromosome loss observed in this protocol, suggesting both a mechanism and strategy for T cell engineering that mitigates this genotoxicity in the clinic.

cell biology↗

bioRxiv2021

Pooled screening of CAR T cells identifies non-native signaling domains for next- generation immunotherapies

Chimeric antigen receptors (CARs) repurpose natural signaling components to retarget T cells to refractory cancers, but have shown limited efficacy against solid tumors. Here, we introduce CAR Pooling, a multiplexed approach to rapidly identify CAR designs with clinical potential. Forty CARs with diverse immune costimulatory domains were assessed in pooled assays for their ability to stimulate critical T cell effector functions during repetitive stimulation that mimics long-term tumor antigen exposure. Several non-native domains from the TNF receptor family exhibited enhanced proliferation (CD40) or cytotoxicity (BAFF-R and TACI) relative to clinical benchmarks, and fell into distinct states of memory, cytotoxicity, and metabolism. BAFF-R CAR T cells were enriched for a highly cytotoxic and NK-cell-like innate phenotype previously associated with positive clinical outcomes. CAR Pooling enables efficient exploration of how CAR design affects cell activity and can be applied to optimize receptors across a range of applications and cell types.

immunology↗

bioRxiv2020

SCITO-seq: single-cell combinatorial indexed cytometry sequencing

The development of DNA-barcoded antibodies to tag cell-surface molecules has enabled the use of droplet-based single cell sequencing (dsc-seq) to profile the surface proteomes of cells. Compared to flow and mass cytometry, the major limitation of current dsc-seq-based workflows is the high cost associated with profiling each cell, thus precluding its use in applications where millions of cells are required. Here, we introduce SCITO-seq, a new workflow that combines combinatorial indexing and commercially available dsc-seq to enable cost-effective cell surface proteomic sequencing of greater than 105 cells per microfluidic reaction. We demonstrate SCITO-seqs feasibility and scalability by profiling mixed species cell lines and mixed human T and B lymphocytes. To further demonstrate its applicability, we show comparable cellular composition estimates in peripheral blood mononuclear cells obtained with SCITO-seq and mass cytometry. SCITO-seq can be extended to include simultaneous profiling of additional modalities such as transcripts and accessible chromatin or tracking of experimental perturbations such as genome edits or extracellular stimuli.

genomics↗

bioRxiv2018

TnClone: high-throughput clonal analysis usingTn5-mediated library construction and de novo assembly

A typical molecular cloning procedure requires Sanger sequencing for validation, which becomes cost-prohibitive and labour-intensive for large-scale clonal analysis of genotype-phenotype studies. Here we present a Tn5-mediated clonal analysis platform TnClone, which uses next-generation sequencing (NGS) to rapidly and cost-effectively analyze a large number of clones. We also developed a user-friendly graphical user interface and have provided general guidelines for conducting validation experiments. Using TnClone, we achieved more than 20-fold cost reduction compared with the cost incurred using conventional Sanger sequencing and detected low-frequency mutant clones (~10%) in mixed samples. We tested our programme and achieved 99.4% sensitivity. Our platform provides rapid turnaround with minimal hands-on time for secondary evaluation as NGS technology continues to evolve.

synthetic biology↗

bioRxiv2018

Lineage tracing using a Cas9-deaminase barcoding system targeting endogenous L1 elements

Determining cell lineage and function is critical to understanding human physiology and pathology. Although advances in lineage tracing methods have provided new insight into cell fate, defining cellular diversity at the mammalian level remains a challenge. Here, we developed a genome editing strategy using a cytidine deaminase fused with inactive Cas9 (dCas9) to specifically target endogenous interspersed repeat regions in mammalian cells. The resulting mutation patterns served as a genetic barcode, which was induced by targeted mutagenesis with single-guide RNA (sgRNA), leveraging substitution events, and subsequent read out by a single primer pair. By analyzing interspersed mutation signatures, we show the accurate reconstruction of cell lineage using both bulk cell and single-cell data. We envision that our genetic barcode system will enable fine-resolution mapping of organismal development in healthy and diseased mammalian states.

bioengineering↗