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

Abstract

Infection with the food-borne liver fluke Opisthorchis viverrini 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 (Ov-GRN-1) in pre-malignant lesions by undertaking programmed CRISPR/Cas9 knockout of the Ov-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 Ov-GRN-1. Gene editing resulted in rapid depletion of Ov-GRN-1 transcripts and the encoded Ov-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 Ov-GRN-1 in virulence morbidity during opisthorchiasis.

Abstract

In the rural regions alongside the Mekong River in South East Asia, traditional cuisines often use uncooked or under cooked fish, many of which carry a worm known as Opisthorchis viverrini. Once inside the body, this parasite settles in the human liver, causing a tropical disease known as liver fluke infection. Out of the 10 million people affected by O. viverrini, thousands will also develop a type of liver cancer that is triggered by the presence of the worm. In particular, the parasite secretes a protein known as granulin that may encourage certain liver cells to multiply, potentially raising the risk for cancer.

A gene editing technique called CRISPR/Cas9 allows scientist to precisely target and then deactivate the genetic information a cell needs to produce a given protein. While the tool has been used in other species before, it was unknown if it could be applied to O. viverrini. Here, Arunsan et al. harnessed CRISPR/Cas9 to deactivate the gene that codes for granulin and create parasites that can only produce very little of the protein.

Hamsters infected with the gene-edited worms had fewer symptoms of liver fluke infection compared to those carrying normal O. viverrini. The animals with parasites that cannot produce granulin also had fewer changes to the liver that are associated with cancer. These findings confirm that granulin has a role in promoting liver fluke infection and liver cancer.

Alongside this work, Ittiprasert et al. used CRISPR/Cas9 to inactivate a gene in a species of worm that causes a human disease called schistosomiasis. Together, these findings demonstrate for the first time that the gene editing method can be adapted for use in parasitic worms, which are a major public health problem in tropical climates. This tool should help scientists understand how the parasites invade and damage our bodies, and provide new ideas for treatment and disease control.