Using CRISPR-Cas9 gene editing, we successfully generated HMGB2-knockout NK-92 cells, which exhibited enhanced activating receptor expression, increased secretion of cytotoxic molecules (granzyme B, perforin) and cytokines (IFN-γ, TNF-α), and significantly improved cytotoxic activity against ESCC cell lines. Here, GZMB is linked to esophageal squamous cell carcinoma.