Targeted mutagenesis in a human-parasitic nematode

Abstract

Parasitic nematodes infect over 1 billion people worldwide and cause some of the most common neglected tropical diseases. Despite their prevalence, our understanding of the biology of parasitic nematodes has been limited by the lack of tools for genetic intervention. In particular, it has not yet been possible to generate targeted gene disruptions and mutant phenotypes in any parasitic nematode. Here, we report the development of a method for introducing CRISPR-Cas9-mediated gene disruptions in the human-parasitic threadworm Strongyloides stercoralis. We disrupted the S. stercoralis twitchin gene unc-22, resulting in nematodes with severe motility defects. Ss-unc-22 mutations were resolved by homology-directed repair when a repair template was provided. Omission of a repair template resulted in deletions at the target locus. Ss-unc-22 mutations were heritable; we passed Ss-unc-22 mutants through a host and successfully recovered mutant progeny. Using a similar approach, we also disrupted the unc-22 gene of the rat-parasitic nematode Strongyloides ratti. Our results demonstrate the applicability of CRISPR-Cas9 to parasitic nematodes, and thereby enable future studies of gene function in these medically relevant but previously genetically intractable parasites.

Author summary

Parasitic worms are a widespread public health burden, yet very little is known about the cellular and molecular mechanisms that contribute to their parasitic lifestyle. One of the major barriers to better understanding these mechanisms is that there are currently no available methods for making targeted gene knockouts in any parasitic worm species. Here, we describe the first mutant phenotype in a parasitic worm resulting from a targeted gene disruption. We applied CRISPR-Cas9-mediated mutagenesis to parasitic worms in the genus Strongyloides and developed a method that overcomes many of the challenges that have previously inhibited generating mutant parasitic worms. We characterize heritable mutant phenotypes and outline a toolkit that will be applicable to many other genes with potential roles in parasitism. Importantly, we developed our method for gene knockouts in a human-parasitic worm. By directly investigating the genes and molecular pathways that enable worms to parasitize humans, we may be able to develop novel anthelmintic therapies or other measures for preventing nematode infections.