Photo‐Enhanced CRISPR/Cas9 System Enables Robust PD‐L1 Gene Disruption in Cancer Cells and Cancer Stem‐Like Cells for Efficient Cancer Immunotherapy

Abstract Blocking immune checkpoint pathways with an antibody or small interfering RNA (siRNA) has become a promising method to reactivate antitumor responses for cancer treatment. However, both blockade strategies achieve only temporary inhibition of these immune checkpoints. Herein, a photoswitched CRISPR/Cas9 system for genomic disruption of the PD‐L1 gene is developed to achieve permanent blockade of the PD‐1/PD‐L1 pathway; this system is constructed by using a photoactivated self‐degradable polyethyleneimine derivative and the plasmid pX330/sgPD‐L1 (expression of the Cas9 protein and single‐guide RNA targeting PD‐L1). Under light irradiation, this photoswitched CRISPR/Cas9 system efficiently genetically disrupts the PD‐L1 gene in not only bulk cancer cells but also cancer stem‐like cells. As a result, the photoswitched CRISPR/Cas9 system significantly increases the infiltration of CD8 + T cells into tumor tissue, leading to effective activation of a T cell‐mediated antitumor response against cancer cells and cancer stem‐like cells. This study provides an alternative strategy to block the PD‐1/PD‐L1 pathway for efficacious immune checkpoint therapy.