Abstract LB043: A compressed genome-wide CROP-Seq screen to dissect the polarization of THP-1-derived macrophages

Abstract CRISPR screens are a powerful discovery engine for biological research and have been extensively used for drug target discovery and validation. Pooled screens are advantageous because they can easily be performed at genome-scale when linked to read-outs such as cellular fitness or viability. However, these types of screens often provide limited insights into complex molecular mechanisms. Arrayed screens, on the other hand, can be combined with information-rich read-outs, but tend to be challenging to scale and costly. Perturb-Seq/CROP-Seq screens, in which the effects of thousands of CRISPR perturbations can be studied using single-cell RNA sequencing (scRNA-Seq), offer an attractive alternative as they combine CRISPR perturbations in a pooled fashion with a high-content readout. Here, we present a genome-wide CROP-Seq screen to identify genes involved in the polarization of THP-1-derived macrophages. M0 macrophages were differentiated into the M1 subtype that secretes proinflammatory cytokines, phagocytizes microbes, and initiates an immune response. We used a compressed CROP-Seq paradigm in which cells were loaded with multiple sgRNAs to increase throughput, and the phenotypic outcomes were subsequently deconvolved. Macrophage polarization was assessed by scRNA-Seq, focusing on a pre-defined signature of 395 mRNAs. Our experiment identified key regulators of M1 polarization, including TLR4, INFGR1, JAK1 and TYK2. In addition, CITE-Seq was incorporated into the screening workflow as macrophages are known to orchestrate immune responses to microenvironmental stimuli sensed by a complex set of surface receptors. Overall, this experiment establishes the feasibility of genome-scale CROP-Seq screens and thus paves the way towards utilizing this technology for unbiased drug target discovery. Citation Format: Daniela Flehberger, Katarina Lalatovic, Sumit Pawar, Henrik Schmidt, Lukas Badertscher, Tilmann Buerckstuemmer. A compressed genome-wide CROP-Seq screen to dissect the polarization of THP-1-derived macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB043.