A Genome-Wide CROP-Seq CRISPRi Screen for Factors of TCR Signaling
At Myllia, the Perturb-seq (CROP-seq) workflow has been adapted to enable genome-scale CRISPRi (CRISPR interference) screens in Jurkat cells at single-cell resolution. The first-of-its-kind genome-scale CRISPRi screen was conducted to verify factors involved in TCR signaling pathways.
T-cell receptor signaling plays a crucial role in the immune response, initiating a cascade of signaling events that regulate T-cell activation, differentiation, and effector functions. Unravelling these T cell-intrinsic molecular factors could potentially unlock new therapeutic targets and strategies for modulating T cell responses in various immune-related disorders.
The Screen
At Myllia, the Perturb-seq (CROP-seq) workflow has been adapted to enable genome-scale CRISPRi (CRISPR interference) screens in Jurkat cells at single-cell resolution. The first-of-its-kind genome-scale CRISPRi screen was conducted to verify factors involved in TCR signaling pathways. In more detail, a guide RNA library targeting 18,595 human genes was utilized for CRISPR-based gene knockdowns in Jurkat cells expressing the dCas9-KRAB fusion endonuclease. In total, one million Jurkat cells were processed for single-cell RNA sequencing allowing transcriptomic readouts of a final list of 374 marker genes involved in TCR signaling.
Results & Conclusion
The bioinformatic analysis confirmed more than 70 known activators and repressors of TCR signaling cascades, hence showcasing the potential of Perturb-seq (CROP-seq) screens to support translational research. The set of 70 genes affected T cell activation, partitioning to 55 activators (whose
knockout led to diminished signaling) and 15 inhibitors (whose knockout led to enhanced signaling).
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