Abstract 5673: Identification of potential therapeutic targets by in vivo CRISPR screening on xenograft models

Abstract Introduction: CRISPR screens have been successfully used to identify targetable genes for anti-cancer research and drug development. Typically, CRISPR screens are conducted in vitro on cell lines, which do not fully capture the influence of the in vivo physiological environment. It is well known that any genetic editing might trigger the adjustment of gene balance to compensate for the modifications in the target cells and tissues. To address the complexity of cellular interactions and the immunological response, in vivo CRISPR screening on a xenograft model is a compelling approach to unravel biological processes where cells are targeted within their natural physiological conditions. Methods: We have conducted a CRISPR screening approach using the human non-small cell lung cancer cell line A549 by knocking out tumor suppressor genes using a library of 3, 188 single guide RNAs (sgRNA). Subsequently, in vivo testing was carried out with the genetically edited A549 cells with or without treatment with the kinase inhibitor Trametinib. At the study endpoint, tumor samples were isolated and analyzed. Amplicon sequencing was used to capture residual sgRNA. MAGeCK was used to calculate the gene enrichment score. Results: After CRISPR editing, 37 sgRNA corresponding to 20 genes were captured in the edited A549 cell line. In the vehicle-treated A549 xenograft tumors, 45 sgRNA corresponding to 27 genes were captured, of which 17 genes were overlapping with engineered A549 cell culture, 8 upregulated and 9 downregulated. In comparison, in the A549 xenograft tumors of the Trametinib treated group, 38 sgRNA corresponding to 23 genes were discovered, with 15 overlapping genes, 9 upregulated and 6 downregulated. The change in expression levels was abundant. Of the 3, 188 sgRNA, the majority of sgRNAs were filtered out in the A549 cell line, under in vivo evolution and the influence of Trametinib treatment. We believe that the pathways related to this small set of identified genes may reveal potential new targets for anti-cancer research and drug development. Conclusion: The in vivo CRISPR approach offers a valuable and translational tool for discovering novel targets in cancer research, providing insights for new anti-cancer therapies. Citation Format: Ce Zhong, Lu Bai, Tongtong Wang, Yiying Shao, Lingyun Zhang, Lu Zhang, Jingqi Huang. Identification of potential therapeutic targets by in vivo CRISPR screening on xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5673.