Abstract
Background. At least 250 million people worldwide suffer from schistosomiasis, caused by Schistosoma worms. Genome sequences for several Schistosoma species are available, including a high-quality annotated reference for Schistosoma mansoni. 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 S. mansoni, to produce somatic mutations in the omega-1 ( ω1) gene. Methods. We employed CRISPR-Cas9 to introduce somatic mutations in a second gene, SULT-OR, a sulfotransferase expressed in the parasitic stages of S. mansoni, in which mutations confer resistance to the drug oxamniquine. A 262-bp PCR product spanning the region targeted by the gRNA against SULT-OR was amplified, and mutations identified in it by high-throughput sequencing. Results. 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 SULT-OR transcription, or leading to mRNA degradation via the nonsense-mediated mRNA decay pathway. However, no SULT-OR 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 SULT-OR. Conclusions. 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 SULT-OR gene to derive an oxamniquine-resistant stable transgenic line.
Highlights
Schistosomiasis is a major Neglected Tropical Disease (NTD) affecting more than 250 million people worldwide [1]
SULT-OR belongs to a gene family that has expanded in trematodes [37], suggesting it may play an important role in clade-specific biology
Genome editing mediated by CRISPR-Cas has been recently applied to S. mansoni to knock out the egg-specific gene omega-1 (ω1) [21]
Summary
Schistosomiasis is a major Neglected Tropical Disease (NTD) affecting more than 250 million people worldwide [1]. S. mansoni and S. japonicum are the agents of hepato-intestinal schistosomiasis manifested by abdominal pain, liver inflammation and fibrosis that leads to portal hypertension. Infection with S. haematobium, agent of urogenital schistosomiasis, is associated with infertility, haematuria, kidney pathology and squamous cell carcinoma of the bladder. All forms of schistosomiasis are associated with systemic morbidities that include malnutrition, anaemia, physical and/or cognitive impairment and stunted development in children [2]. Praziquantel is the single effective drug to treat the infection, and is employed in mass drug administration programmes across endemic areas, which could eventually lead to drug resistance emerging [3]. High-throughput datasets, including high quality reference genomes for the three main species of schistosomes [5,6,7], have been generated. A thorough transcriptome analysis during the parasite’s intra-mammalian development [8], and the identification of different cell types by single-cell RNA sequencing of various life cycle stages [9, 10] represent significant steps towards deciphering cell fate and pathways involved in parasite development and host-parasite interactions
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