Single-Cell Signaling Network Profiling of Pooled CRISPR Screens by Mass Cytometry

/Abstract Deciphering how gene perturbations reshape cellular signaling networks is essential for un- derstanding disease mechanisms and discovering therapeutic targets. However, current pooled CRISPR screening platforms lack the scalability, throughput, and protein-level resolution needed to capture complex cellular responses, especially those governed by post-translational modifications. This collaborative project between Dr. Xiaokang Lun’s lab at the University of Minnesota and Dr. Jellert Gaublomme’s lab at Columbia University seeks to overcome these limitations by developing CRISPRmap-CyTOF, a novel single-cell screening platform that integrates high-complexity pooled CRISPR perturbations with high-dimensional protein-level profiling using mass cytometry. The platform builds on the CRISPRmap combinatorial barcoding strategy (developed by the Gaublomme lab) and a photocrosslinking-based DNA stabilization method (developed by the Lun lab), combining mass cytometry-based signaling network analysis to simultaneously decode thousands of gene perturbations and quantify ∼30 protein or phospho-epitope markers in individual cells. This one-step, high-throughput readout enables proteome-resolved CRISPR screens at single-cell resolution across millions of cells. In Aim 1, we will re-engineer CRISPRmap for compatibility with mass cytometry, optimizing probe chemistry, staining conditions, and barcode decoding algorithms to enable high-fidelity linkage between gene perturbation and signaling phenotypes. In Aim 2, we will apply CRISPRmap-CyTOF to systematically profile early and late signaling responses to a kinome-scale CRISPR knockout library in chronic myeloid leukemia (CML) cells, with and without tyrosine kinase inhibitor (TKI) treatment. These experiments will identify kinases that drive CML progression, uncover novel TKI resistance mechanisms, and generate integrative signaling biomarkers predictive of disease outcome. Together, this work will deliver a transformative technology for functional genomics and a mecha- nistic framework for dissecting therapy resistance in cancer. Project Number: 1R21CA313195-01 | Fiscal Year: 2026 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: Xiaokang Lun (+1 co-PI) | Institution: UNIVERSITY OF MINNESOTA, MINNEAPOLIS, MN | Award Amount: $406,325 | Activity Code: R21 | Study Section: Cellular and Molecular Technologies Study Section[CMT] View on NIH RePORTER: https://reporter.nih.gov/project-details/11354329