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At least 250 million people worldwide suffer from schistosomiasis, caused by  worms. Genome sequences for several  species are available, including a high-quality annotated reference for . 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 , to produce somatic mutations in the  ( ) gene.  We employed CRISPR-Cas9 to introduce somatic mutations in a second gene, , a sulfotransferase expressed in the parasitic stages of , in which mutations confer resistance to the drug oxamniquine. A 262-bp PCR product spanning the region targeted by the gRNA against  was amplified, and mutations identified in it by high-throughput sequencing.  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  transcription, or leading to mRNA degradation  the nonsense-mediated mRNA decay pathway. However, no  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 .  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  gene to derive an oxamniquine-resistant stable transgenic line.

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