Abstract A17: Generation and comparison of CRISPR/Cas9 and Cre-mediated genetically engineered mouse models of sarcoma

Abstract

Genetically engineered mouse models (GEMMs) that employ site-specific recombinase (SSR) technology are important tools for cancer research, and recently the CRISPR/Cas9 system has been increasingly utilized to model cancer in mice. Here, we used CRISPR/Cas9 to generate two primary mouse models of sarcoma, undifferentiated pleomorphic sarcoma (UPS) in a GEMM, and malignant peripheral nerve sheath tumor (MPNST) in wild-type mice, to demonstrate the versatility of the system to generate multiple soft-tissue sarcoma subtypes. Because CRISPR technology is becoming more prevalent in cancer modeling, it is critical to thoroughly evaluate if these models are indeed comparable as tools to study cancer biology compared to conventional GEMMs initiated by recombinase technology. We used two Kras-driven sarcoma models of UPS generated with either Cre recombinase technology or CRISPR/Cas9 technology and compared the mutational profiles, histology, and growth kinetics of these models. KrasLSL-G12D/+; Rosa26LSL-Cas9-EGFP/+ (KC) mice received intramuscular delivery of an adenovirus expressing Cre recombinase and a single guide RNA (sgRNA) targeting Trp53. Cre-mediated expression of oncogenic Kras and Cas9, in combination with CRISPR/Cas9-mediated knockout of Trp53, was sufficient to generate primary soft-tissue sarcomas. Compared to the Cre/loxP model, we determined that sarcomas generated with CRISPR/Cas9 had similar growth kinetics, histology, copy number variation, and mutational load as assessed by whole-exome sequencing. We also demonstrated that off-target mutations in the sarcomas initiated by the Cas9 endonuclease were rare in tumors. Finally, we analyzed the Cas9-mediated indels present in tumors as genetic barcodes, which will enable future studies of tumor heterogeneity and clonality. These results show that sarcomas generated with CRISPR/Cas9 technology are similar to sarcomas generated with conventional modeling techniques. Ultimately this work corroborates CRISPR/Cas9-generated mouse models with traditional GEMMs phenotypically and genotypically, and expands the range of sarcoma mouse models available for research. Citation Format: Jianguo Huang, Mark Chen, Melodi Javid Whitley, Hsuan-Cheng Kuo, Eric S. Xu, Andrea Walens, Yvonne M. Mowery, David Van Mater, William C. Eward, Diana M. Cardona, Lixia Luo, Yan Ma, Omar M. Lopez, Christopher E. Nelson, Jacqueline N. Robinson-Hamm, Anupama Reddy, Sandeep S. Dave, Charles A. Gersbach, Rebecca D. Dodd, David G. Kirsch. Generation and comparison of CRISPR/Cas9 and Cre-mediated genetically engineered mouse models of sarcoma [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr A17.