Multiplexed CRISPR/Cas9 Editing of Tumor Suppressor Genes Recapitulates Molecular and Morphological Features of High-Risk Endometrial Cancer

High-risk endometrial cancers (EC), such as uterine carcinosarcomas (UCS) and serous endometrial intraepithelial carcinoma (SEIC), are characterized by frequent mutations in tumor suppressor genes (TSGs) and poor clinical outcomes. Traditional genetically engineered mouse models are limited in flexibility and scalability to study the cooperative effects of multiple TSG alterations. Here, we use a multiplexed CRISPR/Cas9-based approach to simultaneously edit the top ten TSGs commonly mutated in high-risk EC directly in the mouse endometrium via intrauterine electroporation. Using rolling circle amplification (RCA) and next-generation sequencing, we demonstrate that this method induces targeted gene editing in a mosaic manner, mimicking tumor heterogeneity. We demonstrate that this approach generates histologically and molecularly faithful models of SEIC and UCS. Importantly, some edited tissues remained histologically normal, emphasizing the complex multistep nature of endometrial tumorigenesis. These CRISPR/Cas9-generated murine models serve as robust platforms to dissect the molecular underpinnings of high-risk endometrial cancer and to accelerate preclinical evaluation of novel therapeutic strategies.