Parasitic nematodes infect over 1.5 billion people worldwide and cause some of the most common neglected tropical diseases. Despite their prevalence, the development of novel strategies to prevent nematode infections has been limited by the genetic intractability of these parasites. Here, we report the use of CRISPR-Cas9 for generating targeted gene disruptions in the human-parasitic threadworm Strongyloides stercoralis. We used CRISPR-Cas9 to disrupt the S. stercoralis ortholog of the C. elegans
unc-22 gene, which encodes twitchin, a large intracellular muscle protein homologous to mammalian connectin. In C. elegans,
unc-22 mutants have an uncoordinated (unc) phenotype characterized by decreased motility and body twitching; the twitching phenotype is enhanced upon exposure to acetylcholine receptor agonists such as nicotine. We generated Strongyloides-specific CRISPR-Cas9 constructs targeting
Ss-unc-22 and microinjected the gonads of S. stercoralis free-living adults. A subset of F1 progeny displayed characteristic unc phenotypes, including abnormal swimming and crawling, and severe twitching when exposed to nicotine. We exploited the
Ss-unc-22 twitching phenotype in nicotine to optimize CRISPR-Cas9 conditions in Strongyloides. We tested plasmid-based and ribonucleoprotein complex delivery of CRISPR constructs at three different
Ss-unc-22 target sites. Both plasmid and RNP delivery methods were effective and all three target sites tested produced
Ss-unc-22 F1 mutant progeny. CRISPR-Cas9-induced double-strand breaks were robustly resolved by homology-directed repair (HDR) when an appropriate repair template was provided. The presence of an HDR template significantly improved targeting efficiency and occasionally produced
Ss-unc-22 homozygous knockouts in the F1 generation. Interestingly, in the absence of HDR, we found no evidence for indels at
Ss-unc-22 targets tested, but instead observed large deletions (>1 kb) surrounding the target locus. We are now investigating the mechanism of double-strand break repair in the absence of an HDR template. Finally, we demonstrated that
Ss-unc-22 mutations are heritable by passing mutant F1 progeny through a laboratory host and collecting F2/F3 nematodes with
Ss-unc-22 phenotypes. Our results pave the way for mechanistic studies of gene function in parasitic nematodes, and may enable the development of new strategies for nematode control.