StealTHY: enabling CRISPR-Cas9 screens in immunocompetent hosts to expose concealed metastatic regulators [CRISPR]

CRISPR screens have become standard gene discovery platforms in various contexts, including cancer. Yet, commonly available CRISPR-Cas9 tools are increasingly recognized as unfit for in vivo investigations in immunocompetent contexts, due to the broad immunogenicity of bacterial nucleases and reporters. Here, we show how CRISPR screens with tumor grafts are systematically jeopardized by immunoediting of Cas9-expressing cells in immunocompetent hosts, resulting in iatrogenic clonal dropouts and ultimately compromising target identification. Overall design: Here, we systematically evaluated the immunogenicity of gold standard CRISPR-Cas9 tools in murine and humanized models of metastatic carcinoma, exploring the specific impact of each constitutive transgenic component on the tumor development, clonality, gene expression features and metastasis formation. In addition, we introduce a novel platform designed to elude the combined immunogenicity of ectopic Streptococcus pyogenes Cas9 and bacterial proteins like Puromycin-N-acetyltransferase (PuroR), enabling the implementation of fully autologous in vivo CRISPR screens that provide more accurate insights into cancer-immune system interactions.