In vivo CRISPR Screening Identifying Therapeutic Target to Sensitize Anti-PD-1 Immunotherapy in Non-small Cell Lung Cancer

Although immunotherapy has achieved great success in lung adenocarcinoma (LUAD), only a subset of patients exhibit a favorable response. Leveraging LUAD mono-immunotherapy RNA-sequencing data, we identified differentially expressed genes associated with immunotherapy efficacy by comparing immunotherapy-sensitive and immunotherapy-insensitive patients. These genes were utilized to construct a customized sgRNA library. Two in vivo CRISPR screening models including Lewis lung carcinoma (LLC) and KrasG12D/Trp53-/-(KP) models were developed to identify novel targets regulating immunotherapy efficacy. After transducing the customized sgRNA library, LLC/KP-Cas9 library cells were injected into mice divided into three groups: Rag1-/- C57BL/6 immunodeficient mice treated with IgG antibody, and wild type C57BL/6 immunocompetent mice treated with anti-PD-1 antibody or isotype IgG antibody. Tumors were harvested after 2-4 weeks of treatment, genomic DNA was isolated, and next-generation sequencing (NGS) was performed to analyze sgRNA distribution. By comparing the sgRNAs detected in wild-type C57BL/6 immunocompetent mice treated with anti-PD-1 antibody versus isotype IgG, potential targets that regulating immunotherapy efficacy were identified. Lewis lung carcinoma and KrasG12D/Trp53-/- mouse models were used for in-vivo CRISPR screen. The mice were divided into 3 groups randomly, including Rag1-/- C57BL/6 immunodificient mice with IgG antibody treatment, wild type C57BL/6 immunocompetent mice with treatment of anti-PD-1 antibody or isotype IgG antibody.