Programmed genome editing of the omega-1 ribonuclease of the blood fluke, Schistosoma mansoni

Abstract

CRISPR/Cas9-based genome editing has yet to be reported in species of the Platyhelminthes. We tested this approach by targeting omega-1 (ω1) of Schistosoma mansoni as proof of principle. This secreted ribonuclease is crucial for Th2 polarization and granuloma formation. Schistosome eggs were exposed to Cas9 complexed with guide RNA complementary to ω1 by electroporation or by transduction with lentiviral particles. Some eggs were also transfected with a single stranded donor template. Sequences of amplicons from gene-edited parasites exhibited Cas9-catalyzed mutations including homology directed repaired alleles, and other analyses revealed depletion of ω1 transcripts and the ribonuclease. Gene-edited eggs failed to polarize Th2 cytokine responses in macrophage/T-cell co-cultures, while the volume of pulmonary granulomas surrounding ω1-mutated eggs following tail-vein injection into mice was vastly reduced. Knock-out of ω1 and the diminished levels of these cytokines following exposure showcase the novel application of programmed gene editing for functional genomics in schistosomes.

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

“…The most common deletions extended 34 bases upstream, completely removing the first coding exon (barring a single a base) and shifting the reading frame of the entire coding region ( Figure 1C and Supplementary Figure S7). This would result in the preferential degradation of the mutant mRNAs, as previously suggested to explain the ω 1 knockdown at the mRNA level observed in CRISPR-Cas9-treated eggs [21]. Moreover, any remaining frame-shifted SULT-OR protein is predicted to have lost its ability to convert OXA to its active drug form.…”

“…An interesting question is whether the pattern of CRISPR-Cas-induced mutations varies from gene-to-gene. When targeting the ω 1 gene in the absence of donor molecule, where CRISPR-Cas9-induced mutations presumably occurred by non-homologous end joining (NHEJ) [19], the overall rate of deletions detected in reads from CRISPR-Cas9-treated eggs was not higher than in control eggs [21]. This is consistent with an extremely low rate of NHEJ-mediated deletions in the ω 1 gene in eggs, similar to what we described here for the genome editing of the SULT-OR gene in eggs.…”

“…This is consistent with an extremely low rate of NHEJ-mediated deletions in the ω 1 gene in eggs, similar to what we described here for the genome editing of the SULT-OR gene in eggs. On the other hand, when the ω 1 gene was targeted using CRISPR-Cas9 in the presence of a ssODN donor molecule, rare larger deletions spanning the predicted Cas9 cut site were detected in the CRISPR-Cas9-treated eggs compared to controls [21]. Since this effect was only observed in the presence of the donor molecule, it is tempting to speculate that those large deletions in the ω 1 gene may have been related to the homology directed repair (HDR) mechanism involved in the knock-in of the donor molecule rather than driven by NHEJ [43].…”

“…CRISPR-Cas9 has recently been used to create a heritable gene knock-out line in the parasitic nematode Strongyloides stercoralis [20]. In S. mansoni, the technology has been used to produce mutations in the omega-1 (ω1) gene in somatic cells of the egg [21], and in a related parasitic flatworm, the liver fluke Opisthorchis viverrini, CRISPR-Cas9 mutations have been introduced into the granulin gene in somatic cells of adult worms [22]. Somatic mutations in these two flatworms were associated with dramatic reductions in ω 1 and granulin mRNA levels, respectively, and produced in vitro and in vivo phenotypic effects shedding new light on their functional roles and contributions to pathogenesis [21,22].…”

Liver fluke granulin promotes exosome-mediated crosstalk and cellular microenvironment conducive to cholangiocarcinoma

“…Virions were derived following transfection of human 293T cells producer cells, using FUGENE HD transfection reagent (Promega, Madison, WI), with pLV-huPGRNx2 construct and the MISSION TM lentiviral packaging kit (Sigma-Aldrich), as described (59). Pooled culture supernatants containing pseudotyped virions were collected at 48 to 72 h after transfection of 293T cells, clarified by centrifugation at 500×g for 10 min, and passed through a Millipore 0.45 µm pore membrane (Steriflip-GP, Millipore).…”