Abstract 2194: Rapid interrogation of colon cancer driver genes in vivo using split Cre and CRISPR-Cas9

Abstract Colorectal cancer (CRC) is the second leading cause of cancer-related deaths worldwide, with a five-year survival rate of only 63% with current treatment regimens. While genetically engineered mouse models (GEMMs) have contributed widely to our understanding of the underlying genetics and molecular mechanisms of the disease, major limitations exist. Most models of CRC lack flexibility to initiate focal adenocarcinomas with different genetic perturbations within the native microenvironment of the distal colon. Specifically, no autochthonous model of metastatic CRC compatible with in vivo CRISPR-Cas9 genome engineering has been described. Therefore, we developed a novel split Cre-recombinase system restricting activation of oncogenic KrasG12D and deletion of the tumor suppressor gene (TSG) Trp53 to Lgr5+ stem cells of the colon crypts. Importantly, these events are not only critical drivers of CRC progression and metastasis in mice and humans but can also lead to non-specific transformation of the surrounding colonic stroma. By expressing one half of Cre from the endogenous Lgr5 locus and other half via colonoscopy-guided submucosal injection of lentivirus, we restricted Cre-mediated recombination to enterocytes. Lentivirus was also engineered to express a single guide RNA (sgRNA) against Apc, facilitating tumor initiation via deletion of this critical CRC TSG in mice expressing Cas9. A second sgRNA was included to facilitate rapid interrogation of putative cancer driver genes. As proof-of-principle, we targeted Smad4, a critical suppressor of metastasis in CRC, and observed adenocarcinoma formation and liver metastases with complete recombination and editing of all targeted genes. To prioritize candidate TSGs for future in vivo interrogation, we applied MutSigCV to large human sequencing datasets and identified genes significantly co-mutated with APC and KRAS in CRC. Altogether, this faithful and tractable model of CRC can be used for rapid functional interrogation of TSGs and should accelerate downstream mechanistic and preclinical studies. Citation Format: Daniel Zhang, Peter Westcott, Santiago Naranjo, Haley Hauck, William Rideout, Nate Sacks, Arjun Bhutkar, Justin Patten, Norihiro Goto, Joseph Sedlak, Tyler Jacks. Rapid interrogation of colon cancer driver genes in vivo using split Cre and CRISPR-Cas9 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2194.