Biology subjects

Bourke, S.

Publications and source records attributed to Bourke, S..

3 recordsLinked to original sources

bioRxiv2025

RainBar: Optical Barcoding for Pooled Live-Cell Imaging with Single-Cell Resolution

High-throughput pooled screening has advanced functional genomics, but most existing methods rely on endpoint sequencing and are blind to dynamic, time-resolved phenotypes. We developed RainBar (Rainbow Barcodes), an optical barcoding system that supports pooled live-cell imaging with single-cell resolution. RainBar uses lentiviral co-delivery of spectrally distinct nuclear and cytoplasmic fluorescent proteins to encode up to 64 unique perturbations per well. To mitigate barcode recombination and improve decoding accuracy, we employed single-template viral production, codon diversification, and a ratio-based spectral unmixing pipeline tailored to overlapping fluorophores. An inverted cytoplasmic segmentation approach and multilayer perceptron classifier enabled accurate barcode identification in both arrayed and pooled formats. As a proof of concept, we applied RainBar to dissect NF-{kappa}B signaling dynamics in epithelial cells. Live imaging of RelA translocation uncovered stimulus-specific kinetics: IL-1{beta} triggered rapid recovery, while TNF induced prolonged nuclear localization. In pooled CRISPRi screens, RainBar recovered known NF-{kappa}B regulators (e.g., IL1R1, MYD88, TNFRSF1A) and highlighted additional modulators, including the Ino80 chromatin remodeling complex subunits and KAT2A acetyltransferase. Together, these results position RainBar as a flexible platform for multiplexed, image-based functional genomics, with potential to reveal dynamic signaling architectures across diverse cellular contexts in live cells.

genomics↗

bioRxiv2024

Scalable intracellular delivery via microfluidic vortex shedding enhances the function of chimeric antigen receptor T-cells

Adoptive chimeric antigen receptor T-cell (CAR-T) therapy is transformative and approved for hematologic malignancies. It is also being developed for the treatment of solid tumors, autoimmune disorders, heart disease, and aging. Despite unprecedented clinical outcomes, CAR-T and other engineered cell therapies face a variety of manufacturing and safety challenges. Traditional methods, such as lentivirus transduction and electroporation, result in random integration or cause significant cellular damage, which can limit the safety and efficacy of engineered cell therapies. We present hydroporation as a gentle and effective alternative for intracellular delivery. Hydroporation resulted in 1.7- to 2-fold higher CAR-T yields compared to electroporation with superior cell viability and recovery. Hydroporated cells exhibited rapid proliferation, robust target cell lysis, and increased pro-inflammatory and regulatory cytokine secretion in addition to improved CAR-T yield by day 5 post-transfection. We demonstrate that scaled-up hydroporation can process 5 x 108 cells in less than 10 s, showcasing the platform as a viable solution for high-yield CAR-T manufacturing with the potential for improved therapeutic outcomes.

bioengineering↗

bioRxiv2020

Versatile labeling and detection ofendogenous proteins using tag-assistedsplit enzyme complementation

Recent advances in genome engineering have expanded our capabilities to study proteins in their natural states. In particular, the ease and scalability of knocking-in small peptide tags has enabled high throughput tagging and analysis of endogenous proteins. To improve enrichment capacities and expand the functionality of knock-ins using short tags, we developed the tag-assisted split enzyme complementation (TASEC) approach, which uses two orthogonal small peptide tags and their cognate binders to conditionally drive complementation of a split enzyme upon labeled protein expression. Using this approach, we have engineered and optimized the tag-assisted split HaloTag complementation system (TA-splitHalo) and demonstrated its versatile applications in improving the efficiency of knock-in cell enrichment, detection of protein-protein interaction, and isolation of biallelic gene edited cells through multiplexing.

bioengineering↗