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A novel, high-density CRISPR activation platform for mapping cancer dependencies and resistance pathways ex vivo and in vivo

Diepstraten, S.; Deng, Y.; Potts, M. A.; Heidersbach, A.; Koenig, C.; Dorighi, K.; Tai, L.; Kueh, A.; Whelan, L.; Chang, C.; Brown, F.; Kelly, G.; Fortin, J.-P.; Haley, B.; LaMarca, J.; Herold, M. J.

2025-07-22 cancer biology
10.1101/2025.07.22.666069 bioRxiv
Show abstract

CRISPR activation (CRISPRa) enables precise, locus-specific upregulation of gene expression, offering potential for both ex vivo and in vivo applications. However, the lack of scalable, high-coverage tools has limited its use in comprehensive genetic screens, particularly in murine models. Here, we introduce Partita, a next-generation, whole-genome CRISPRa sgRNA platform designed for unparalleled efficiency and depth in gene activation studies. Partita employs a high-density targeting strategy, deploying 10 sgRNAs per transcription start site, structured into five gene family-specific sub-libraries to maximize transcriptional induction. To demonstrate its capabilities, we performed a series of large-scale screens: an in vitro enrichment/depletion screen in iBMDMs, whole-genome CRISPRa screens in a double-hit lymphoma model to uncover genes driving resistance to pro-apoptotic drugs (venetoclax, nutlin-3a, etoposide), and an in vivo whole-genome screen identifying accelerators of Myc-driven lymphomagenesis. Each experiment revealed both expected and novel regulators of cellular phenotypes, with a high validation rate in secondary assays. By enabling robust, high-throughput gain-of-function screening, Partita unlocks new avenues for functional genomics and expands the toolkit for discovering key drivers of biological processes across diverse research fields.

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