Large CRISPR-Cas-induced deletions in the oxamniquine resistance locus of the human parasite Schistosoma mansoni

Abstract

<ns3:p> <ns3:bold>Background.</ns3:bold> At least 250 million people worldwide suffer from schistosomiasis, caused by <ns3:italic>Schistosoma</ns3:italic> worms. Genome sequences for several <ns3:italic>Schistosoma</ns3:italic> species are available, including a high-quality annotated reference for <ns3:italic>Schistosoma mansoni</ns3:italic> . There is a pressing need to develop a reliable functional toolkit to translate these data into new biological insights and targets for intervention. CRISPR-Cas9 was recently demonstrated for the first time in <ns3:italic>S. mansoni</ns3:italic> , to produce somatic mutations in the <ns3:italic>omega-1</ns3:italic> ( <ns3:italic>ω1</ns3:italic> ) gene. </ns3:p> <ns3:p> <ns3:bold>Methods.</ns3:bold> We employed CRISPR-Cas9 to introduce somatic mutations in a second gene, <ns3:italic>SULT-OR</ns3:italic> , a sulfotransferase expressed in the parasitic stages of <ns3:italic>S. mansoni</ns3:italic> , in which mutations confer resistance to the drug oxamniquine. A 262-bp PCR product spanning the region targeted by the gRNA against <ns3:italic>SULT-OR</ns3:italic> was amplified, and mutations identified in it by high-throughput sequencing. </ns3:p> <ns3:p> <ns3:bold>Results.</ns3:bold> We found that 0.3-2.0% of aligned reads from CRISPR-Cas9-treated adult worms showed deletions spanning the predicted Cas9 cut site, compared to 0.1-0.2% for sporocysts, while deletions were extremely rare in eggs. The most common deletion observed in adults and sporocysts was a 34 bp-deletion directly upstream of the predicted cut site, but rarer deletions reaching as far as 102 bp upstream of the cut site were also detected. The CRISPR-Cas9-induced deletions, if homozygous, are predicted to cause resistance to oxamniquine by producing frameshifts, ablating <ns3:italic>SULT-OR</ns3:italic> transcription, or leading to mRNA degradation <ns3:italic>via</ns3:italic> the nonsense-mediated mRNA decay pathway. However, no <ns3:italic>SULT-OR</ns3:italic> knock down at the mRNA level was observed, presumably because the cells in which CRISPR-Cas9 did induce mutations represented a small fraction of all cells expressing <ns3:italic>SULT-OR</ns3:italic> . </ns3:p> <ns3:p> <ns3:bold>Conclusions.</ns3:bold> Further optimisation of CRISPR-Cas protocols for different developmental stages and particular cell types, including germline cells, will contribute to the generation of a homozygous knock-out in any gene of interest, and in particular the <ns3:italic>SULT-OR</ns3:italic> gene to derive an oxamniquine-resistant stable transgenic line. </ns3:p>

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