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

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.