Nonviral CRISPR/Cas9 mutagenesis for streamlined generation of mouse lung cancer models

Functional analysis in mouse models is necessary to establish the involvement of a set of genetic variations in tumor development. A modeling platform to facilitate and cost- effectively analyze the role of multiple genes in carcinogenesis would be valuable. Here, we present an innovative strategy for lung mutagenesis using CRISPR/Cas9 ribonucleoproteins delivered via cationic polymers. This approach allows the simultaneous inactivation of multiple genes. We validate the effectiveness of this system by targeting a group of tumor suppressor genes, specifically Rb1, Rbl1, Pten, and Trp53, which were chosen for their potential to cause lung tumors, namely small cell lung carcinoma (SCLC). Tumors with histologic and transcriptomic features of human SCLC emerged after intratracheal administration of CRISPR/polymer nanoparticles. These tumors carried loss- of- function mutations in all four tumor suppressor genes at the targeted positions. These findings were reproduced in two different pure genetic backgrounds. We provide a proof of principle for simplified modeling of lung tumorigenesis to facilitate functional testing of potential cancer- related genes.