Gene editing of a carcinogenic liver fluke tetraspanin impairs parasite surface biogenesis and extracellular vesicle uptake by human host cells.

Opisthorchiasis remains a significant public health concern throughout Southeast Asia. The liver fluke Opisthorchis viverrini resides within the biliary tract and chronic infection leads to bile duct cancer, or cholangiocarcinoma. Here, we examined the functions of liver fluke tetraspanins, four-transmembrane domain proteins expressed on the surface of the fluke tegument and extracellular vesicles (EVs) derived from this syncytial surface. We undertook CRISPR-Cas9 gene knockout (KO) of the O. viverrini tetraspanin-2 (Ov-tsp-2) gene and found that Ov-tsp-2-KO flukes had abnormal tegument biogenesis. The tegument of Ov-tsp-2-KO flukes was increasingly vacuolated and fewer EVs were secreted. EVs that were secreted were deficient in Ov-TSP-2 and their uptake by cholangiocytes was diminished. The findings indicate a critical role for Ov-TSP-2 in maintenance of the tegument, EV production and uptake by host target cells, and support the development of this parasite antigen as an anti-infection and anti-cancer vaccine for opisthorchiasis and opisthorchiasis-associated cholangiocarcinoma.