Single-Cell Analysis Guides Organoid CRISPR Screening to Reveal NF1 as a Gatekeeper of Cervical Squamous Malignant Transformation.

Cervical cancer progresses from high-grade squamous intraepithelial lesions driven by persistent HPV infection. Identifying the gatekeepers restraining malignant transformation could reveal strategies to prevent and treat cervical cancer. Here, we integrated single-cell transcriptomics, CRISPR-Cas9 screening, and organoid modeling to dissect this process. Single-cell analysis of patient samples revealed progressive activation of RAS and proliferation programs along epithelial differentiation paths. A focused CRISPR screen in HPV-positive pre-tumoroids identified NF1 as the top suppressor of malignant transition. NF1 loss accelerated carcinogenesis in organoids, compressing a 5~10-year process into 3~6 months, recapitulating basal cell expansion, dedifferentiation, and tumorigenicity. Mechanistically, NF1 loss enhanced RAS-MAPK and PI3K-AKT signaling, promoted proliferative programs, and remodeled chromatin accessibility at AP-1/E2F motifs. Conversely, NF1 restoration in cancer organoids induced apoptosis and suppressed malignant maintenance. AI-guided modeling was used to design a minimal NF1-mimetic peptide that engaged RAS-GTP to inhibit RAS signaling, reduce tumor burden in HPV16-driven mouse models, and restore local immune permissiveness without systemic activation. Together, these findings establish NF1 as a critical constraint on HPV-associated epithelial evolution and provide a time-compressed organoid model of cervical carcinogenesis, offering proof-of-concept for therapeutic RAS pathway interception via NF1 restoration.