Authors

Discussion

Schistosomiasis is a neglected tropical disease affecting >200 million people worldwide. Genome sequences for several Schistosoma species are available, including a high quality annotated reference for Schistosoma mansoni.  There is now an urgent need to develop a functional toolkit to translate these data into new biological insights and targets for intervention. The eggs of S. mansoni can be transduced using mammalian retroviruses.  Transgenes can be stably integrated into chromosomes and transmitted through the germline as stable transgenic lines. However, in the absence of a robust selection protocol for transgenic parasites, transgenic organisms become diluted among non-transformed parasites and eventually lost in successive generations. A sulfotransferase (ST) has been identified as the gene contributing to resistance against Oxamniquine (OXA), an anthelmintic with schistosomicidal activity against S. mansoni. Here, we target the sulfotransferase locus using CRISPR-Cas 9 and introduce transgenes in to the germ line of S. mansoni, to obtain stable transgenic lines resistant to OXA. Sensitivity to OXA was analysed in eggs and sporocysts, the two key developmental stages suitable for development of germline transgenesis, given the high germline to soma cell ratio present in these stages. Although the eggs were resistant to OXA at the tested concentrations, sporocysts displayed a hypermotile phenotype followed by increasing mortality in a concentration-dependent manner. RNAi experiments against ST in sporocysts cultured in the presence of OXA were performed to investigate the presence of a rescue phenotype, i.e. OXA-resistant sporocysts. In addition, we have designed three ST-specific gRNAs and transfected eggs and sporocysts with fluorescently-labelled ribonucleoprotein complex comprising CRISPR-Cas9 and gRNA. From these transfected parasites, we used PCR-amplicon sequencing to  quantify insertions and deletions in the ST locus, and qRT-PCR to determine the extent to which gene expression was perturbed. Donor DNA cassettes for CRISPR-Cas9 mediated Homology Directed Repair knock-in experiments in the ST locus, and retrovirus expressing Cas9 and ST-specific gRNAs are underway. Retroviral-based approaches coupled with genome editing, driven by CRISPR-Cas9, promise exciting new opportunities to explore the functional genomics investigations of this neglected tropical disease parasite.