Abstract 2949: A flexible and efficient approach for single-cell CRISPR perturbation screens combined with targeted RNA-seq expression analysis

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Abstract This study presents a novel approach for performing 10X platform-based targeted single-cell RNA-seq analysis in combination with CRISPR perturbation screens. For the analysis, we constructed a custom sgRNA library focused on a small subset of genes associated with the TNFα response (i.e., NFκB pathway). We compared analysis of two targeted gene expression panels—the first, targeting all protein-coding genes (genome-wide) and the other, targeting a selected set of genes representing key hubs in signaling pathway modules. Narrowing down the analyzed gene set enhanced the efficiency of single-cell RNA-seq by reducing the associated next-generation sequencing (NGS) depth required for readout, and significantly streamlining data analysis. Comparing the analysis of the expression panels targeting all protein-coding genes with the smaller pathway-targeted set, we were able to assess the general flexibility of the novel approach for single-cell CRISPR perturbation screens. We evaluated the specific tradeoffs with regard to the size of the CRISPR library, the size of the panel of genes targeted for expression analysis, and NGS sequencing depth. This flexible, targeted approach not only refines the precision of RNA-seq but also provides a more cost-effective solution for comprehensive genomic analyses. Citation Format: Donato Tedesco, Tianbing Liu, Mikhail Makhanov, Dongfang Hu, Nadya Isachenko, Paul Diehl, Alex Chenchik. A flexible and efficient approach for single-cell CRISPR perturbation screens combined with targeted RNA-seq expression analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2949.