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• W4366716872 abstract "Abstract CROP-seq/Perturb-seq screens combine CRISPR perturbation with single-cell RNA sequencing. In brief, 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 costs for single-cell library preparation and next generation sequencing are high. Here, we aimed to conduct a genome-scale CRISPR screen within a reasonable budget. We chose Jurkat T cells for this experiment and focused on T cell activation because signaling pathways in these cells are well understood and we could utilize the published knowledge to benchmark the performance of our platform. To accomplish this, we utilized a CRISPR interference setup in which multiple sgRNAs can be delivered to the same cell, thus “squeezing” more perturbations into one cell. We targeted 18.595 human genes with four sgRNAs per gene and delivered this genome-scale library to Jurkat cells harboring dCas9-KRAB. Cells were stimulated with anti-TCR and anti-CD28 antibodies for 24 hours to activate T cell signaling. Following that, we processed 1.000.000 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 selected set of 374 transcripts and submitted the corresponding library for NGS on one NovaSeq S4 flowcell. First, we confirmed that the perturbation of 374 marker genes by CRISPR interference led to the downregulation of the cognate targets, suggesting that the CRISPR perturbation workflow is functional. Then, we assessed whether T cell activation could be recapitulated from the chosen markers and found that activated Jurkat T cells can be distinguished from their unactivated counterparts using the signature in question. 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). Of note, our screen recovered key signaling nodes that are proximal to the TCR and have been well described in the literature, including LAT, LCK, ZAP70, CD3E, ITK, RASGRP1 and VAV1. Overall, the screen presented here will catalyse a paradigm shift for CROP-seq/Perturb-seq type CRISPR screens towards genome scale. Citation Format: Anke Loregger, Nicole Untermoser, Anatoly Vasilyev, Adam Krejci, Henrik Schmidt, Tilmann Bürckstümmer. A genome-scale CROP-seq screen reveals mediators of T cell signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB249." @default.
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• W4366716872 date "2023-04-14" @default.
• W4366716872 modified "2023-10-06" @default.
• W4366716872 title "Abstract LB249: A genome-scale CROP-seq screen reveals mediators of T cell signaling" @default.
• W4366716872 doi "https://doi.org/10.1158/1538-7445.am2023-lb249" @default.
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