Functional CRISPR Screens Define Genetic Drivers for Cancer Transformation and Progression from Non-Cancerous Cells

Tumor initiation and metastatic progression are driven by context-dependent genetic alterations that disrupt tumor suppressor pathways, metabolic homeostasis, and signaling networks. However, the initial drivers that transform normal cells into malignant ones and their context dependency remain elusive. To address this, we aimed to systematically identify and characterize these drivers across cancer types, species, and microenvironments. We constructed customized clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) knockout (KO) libraries targeting high-frequency mutated and downregulated genes associated with liver hepatocellular carcinoma (LIHC) and breast carcinoma (BRCA) and conducted parallel functional screens in non-cancerous mouse and human fibroblast cell lines under two-dimensional (2D), three-dimensional (3D), and in vivo conditions. Strikingly, TP53 and NF1 emerged as pan-context drivers consistently enriched across immortalization, tumorigenesis, and metastasis in both LIHC and BRCA settings, while most other identified drivers were largely species-, tissue-, and microenvironment-specific with limited cross-model overlap. Despite this heterogeneity, all drivers converge on core pathways including epigenetic regulation, metabolic reprogramming, and growth factor signaling. Unlike prior studies on established cancer cells, this work defines the genetic barriers restricting the malignant transformation of primary normal cells, offering a new framework for early cancer evolution.