An Optimized Platform Overcomes Excessive Tumor Immune Rejection Induced by the CRISPR/Cas9 Lentiviral System.

UNLABELLED: The clustered regularly interspaced short palindromic repeats/Cas9 lentiviral system has emerged as a powerful tool for gene knockout in cancer immunology research. However, the persistent expression of exogenous elements, such as Cas9 and resistance markers, often triggers excessive tumor immune rejection. This can lead to prolonged experimental timelines, increased data variability, biased outcomes, and even experimental failures. To address this challenge, several strategies have been investigated that offer partial solutions, but they have failed so far to comprehensively resolve the issue. In response, we developed the v2-Blast-lox2272 (VL)-adenovirus expressing Cre recombinase (AdCre) system, a strategy that enables efficient excision of exogenous expression elements following gene knockout. The VL-AdCre system effectively reduced tumor immune rejection in allograft models, streamlined experimental workflows, and improved the reliability of research outcomes. Overall, this optimized clustered regularly interspaced short palindromic repeats/Cas9 lentiviral gene knockout system offers a robust and practical solution for studying gene functions in vivo and advancing immunotherapeutic strategies.

SIGNIFICANCE: The VL-AdCre system enables effective removal of exogenous components following gene knockout, thus avoiding tumor immune rejection and enhancing the accuracy and reproducibility of in vivo cancer immunology research.