Immunogen-free CRISPR platform reveals metastatic drivers

Standard CRISPR-CAS9 screening approaches in cancer research face a critical limitation: the bacterial Cas9 nuclease and xenogeneic reporter proteins such as GFP or puromycin resistance provoke T cell-mediated immune responses when expressed in immunocompetent hosts. The researchers demonstrated that these responses lead to widespread clonal dropout and skewed sgRNA representation across multiple syngeneic carcinoma models, thereby fundamentally compromising target identification in metastasis screens.

To address this, they developed StealTHY, which combines transient Cas9 delivery via apoprotein transfection with autologous Thy1 surface reporters that evade immune recognition. A self-targeting "hara-kiri" selection system utilising truncated CD8 enables the rapid isolation of edited cells prior to transplantation.

When applied to screens in both syngeneic mice and humanised patient-derived xenograft models, StealTHY preserved natural clonal architecture and revealed metastatic regulators missed by conventional approaches – including receptor tyrosine kinases, TGF-β pathway members and notably the developmental AMH-AMHR2 signalling axis.

Therapeutic blockade of this pathway using a dominant-negative AMHR2 variant triggered tumour regression specifically in immunocompetent hosts, validating its immune-dependent pro-metastatic function. Gene expression signatures derived from StealTHY hits correlated with clinical outcomes across multiple cancer types in TCGA cohorts.

The study was led by Massimo Saini and Nicola Aceto at Swiss Federal Institute of Technology (ETH) Zurich, Switzerland. It was published online in Cell on 7 November 2025.