(1195-D) A genome-scale CROP-Seq screen reveals mediators of T cell signaling
Tuesday, February 6, 2024
2:00 PM – 3:00 PM EST
Location: Exhibit Halls AB
Abstract: CRISPR screens have become the primary discovery engine in modern biology and are widely used to uncover novel targets in immuno-oncology. However, these genetic screens are usually coupled to rather simplistic read-outs such as cell fitness. In contrast, CROP-Seq (or “CRISPR Droplet” Sequencing) screens combine CRISPR perturbation with single-cell transcriptomics, enabling high-content phenotyping at single-cell resolution. Briefly, cells are perturbed with a pooled sgRNA library and transcriptomic profiles of each cell are recorded using conventional single-cell RNA sequencing platforms. Currently, the scale of these screens is limited to the perturbation of a couple of hundred genes, possibly up to 1.000 genes, because of increased costs for single-cell library preparation and next generation sequencing (NGS).
At Myllia, we have built a platform allowing high-content CROP-Seq screens in both CRISPR-ready cancer cell lines as well as primary immune cells. Using CRISPR KO, CRISPR interference or CRISPR activation workflows, we can help accelerate the identification of target genes and regulatory networks involved in immuno-oncology.
Here, we present a first-of-its-kind genome-scale CROP-Seq CRISPR interference screen in Jurkat cells expressing dCas9-KRAB to study TCR signaling. In an attempt of “squeezing” more perturbations into one cell we performed the screen in as little as one million cells and within a reasonable budget. We focused on TCR-mediated activation of Jurkat T cells as the signaling pathways are well understood and we could utilize published knowledge to benchmark the performance of our single-cell CRISPR screening platform. We delivered multiple sgRNAs per cell and targeted 18.595 human genes with 4 sgRNAs per gene. Cells were stimulated with anti-TCR and anti-CD28 antibodies for 24 hours to activate T cell signaling, and we processed about one million cells in one go, using the Chromium X platform that has recently been launched by 10X Genomics. Following single-cell library preparation, we amplified a set of 374 transcripts and submitted the library for NGS.
We confirmed that the perturbation of 374 marker genes by CRISPR interference led to the downregulation of the cognate targets, suggesting a functional CRISPR perturbation workflow. Finally, we assessed the phenotypes of gene knockouts introduced at genome-scale: Of the 18.595 genes perturbed, a 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).
Overall, the advanced CROP-Seq platform combining large-scale perturbations and targeted sequencing (TA) read-outs will catalyze a paradigm shift for CROP-Seq enabling high-throughput functional genomic screens that support the validation of drug targets in autoimmunity, inflammation and immuno-oncology.