During the last decade, there has been notable progress in cancer immunotherapy, including promising clinical success of immune checkpoint inhibitors. Moreover, there is an increasing number of new potential targets for cancer immunotherapy that are currently being developed both as monotherapy or in combination. However, the lack of durable clinical responses due in part to the resistance mechanisms that tumors exhibit in a significant proportion of patients urges novel approaches to find the right therapeutic strategies. The understanding of such tumor evasion mechanisms and the complex interactions that take place in the tumor microenvironment is essential to obtain successful treatments. Functional genomics has emerged as a powerful tool that can help to reveal some of these unknown processes, with CRISPR-Cas9 technology the most prominent example. However, in vivo genetic screens require stable expression of several components that in most cases are not derived from murine cells, which represents a major obstacle in the context of cancer immunology. The inoculation of engineered tumor cell lines in immunocompetent mice results in either tumor rejection or an aberrant response to immunotherapy, making the experiments technically impossible in the first case or providing inconsistent results in the latter. We have designed new vector strategies to overcome the altered immunogenicity of Cas9-expressing tumor cell lines using the Cre-lox system. Expression and effective removal of Cas9 and vector components are monitored using fluorescent reporter proteins. Both genome-editing capacity and normal in vivo behavior of all tested cell lines remain largely unaltered using this novel strategy. This technology represents a major advance for preclinical functional genomics, allowing studies in fully competent animals looking for better combination treatments and resistance mechanism in particular cancer models, and ultimately will help in the design of clinical trials.

Citation Format: Juan Dubrot, Robert T. Manguso, Sarah Kate Lane-Reticker, Austin Ayer, Emily Kessler, Clara Wolfe, Animesh Mahapatra. Novel CRISPR/Cas9 vectors for in vivo tumor functional genomics [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B10.