Programmed knockout mutation of liver fluke granulin attenuates virulence of infection-induced hepatobiliary morbidity.

Infection with the food-borne liver fluke is the principal risk factor (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2012) for cholangiocarcinoma (CCA) in the Lower Mekong River Basin countries including Thailand, Lao PDR, Vietnam and Cambodia. We exploited this link to explore the role of the secreted growth factor termed liver fluke granulin (GRN-1) in pre-malignant lesions by undertaking programmed CRISPR/Cas9 knockout of the GRN-1 gene from the liver fluke genome. Deep sequencing of amplicon libraries from genomic DNA of gene-edited parasites revealed Cas9-catalyzed mutations within GRN-1. Gene editing resulted in rapid depletion of GRN-1 transcripts and the encoded GRN-1 protein. Gene-edited parasites colonized the biliary tract of hamsters and developed into adult flukes, but the infection resulted in reduced pathology as evidenced by attenuated biliary hyperplasia and fibrosis. Not only does this report pioneer programmed gene-editing in parasitic flatworms, but also the striking, clinically-relevant pathophysiological phenotype confirms the role for GRN-1 in virulence morbidity during opisthorchiasis.