Gene editing in the nematode parasite using extracellular vesicles to deliver active Cas9/guide RNA complexes.

Despite recent advances, animal-parasitic nematodes have thus far been largely refractory to genetic manipulation. We describe here a new approach providing proof of principle that CRISPR/Cas9-mediated gene editing of parasitic nematodes is achievable using vesicular stomatitis virus glycoprotein-pseudotyped extracellular vesicles for the delivery of Cas9-single guide ribonucleoprotein complexes. We demonstrate that extracellular vesicle-delivered ribonucleoproteins can be used to disrupt a secreted deoxyribonuclease in . Introduction of a repair template encoding multiple stop codons led to measurable reduction in expression of the targeted gene. Altered transcripts corresponding to the edited locus were detected by RT-PCR, demonstrating that vesicles can access cells of tissues actively expressing the gene of interest. These data provide evidence that this technique can be employed for targeted gene editing in , making this species genetically tractable for the first time, although further refinement will be necessary for routine and robust interrogation of gene function.