CRISPR/Cas9‐mediated genome editing shows cogent potential for the genetic modification of helminth parasites. We report successful gene knock‐in (KI) into the genome of the egg of Schistosoma mansoni by combining CRISPR/Cas9 with single‐stranded oligodeoxynucleotides (ssODNs). We edited the acetylcholinesterase (AChE) gene of S. mansoni targeting two guide RNAs (gRNAs), X5 and X7, located on exon 5 and exon 7 of Smp_154600, respectively. Eggs recovered from livers of experimentally infected mice were transfected by electroporation with a CRISPR/Cas9‐vector encoding gRNA X5 or X7 combining with/ without a ssODN donor. Next generation sequencing analysis of reads of amplicon libraries spanning targeted regions revealed that the major modifications induced by CRISPR/Cas9 in the eggs were generated by homology directed repair (HDR). Furthermore, soluble egg antigen from AChE‐edited eggs exhibited markedly reduced AChE activity, indicative that programed Cas9 cleavage mutated the AChE gene. Following injection of AChE‐edited schistosome eggs into the tail veins of mice, an significantly enhanced Th2 response involving IL‐4, ‐5, ‐10, and‐13 was detected in lung cells and splenocytes in mice injected with X5‐KI eggs in comparison to control mice injected with unmutated eggs. A Th2‐predominant response, with increased levels of IL‐4, ‐13, and GATA3, also was induced by X5 KI eggs in small intestine‐draining mesenteric lymph node cells when the gene‐edited eggs were introduced into the subserosa of the ileum of the mice. These findings confirmed the potential and the utility of CRISPR/Cas9‐mediated genome editing for functional genomics in schistosomes.

To further investigate the importance of Schistosoma japonicum acetylcholinesterase (SjAChE) in cholinergic signaling for parasite growth and development, we used RNA interference (RNAi) to knock-down its expression in adults and eggs in vitro. This resulted in its reduced transcription but also expression of other important genes involved both in cholinergic signaling and glucose uptake were impacted substantially. Significant decreases in AChE protein expression, AChE enzymatic activity, and glucose uptake were observed in the SjAChE-knockdown parasites compared with luciferase controls. In vaccine/challenge experiments, we found that immunization of mice with recombinant SjAChE (rSjAChE) expressed in Escherichia coli elicited reductions in male worm numbers (33%), liver granuloma density (41%), and reduced numbers of mature intestinal eggs (73%) in the vaccinated group compared with the control group. These results indicate AChE plays an important role in the metabolism of male worms, and impacts indirectly on female fecundity leading to increased numbers of immature eggs being released and reduced sizes of liver granulomas. Furthermore, cytokine analysis showed that immunization of mice with rSjAChE elicited a predominantly Th1-type immune response characterized by increased production of IFNγ in splenic CD4+ T cells of vaccinated mice. The study confirms the potential of SjAChE as a vaccine/drug candidate against zoonotic schistosomiasis japonica.

AbstractCRISPR/Cas9-mediated genome editing shows cogent potential for the genetic modification of helminth parasites. Here we report successful gene knock-in (KI) into the genome of the egg of Schistosoma mansoni by combining CRISPR/Cas9 with single-stranded oligodeoxynucleotides (ssODNs). We edited the acetylcholinesterase (AChE) gene of S. mansoni targeting two guide RNAs (gRNAs), X5 and X7, located on exon 5 and exon 7 of Smp_154600, respectively. A CRISPR/Cas9-vector encoding gRNA X5 or X7 was introduced by electroporation into eggs recovered from livers of experimentally infected mice. Simultaneously, eggs were transfected with a ssODN donor encoding a stop codon in all six frames, flanked by 50 nt-long 5’- and 3’-homology arms matching the predicted Cas9-catalyzed double stranded break at X5 or X7. Next generation sequencing analysis of reads of amplicon libraries spanning targeted regions revealed that the major modifications induced by CRISPR/Cas9 in the eggs were generated by homology directed repair (HDR). Furthermore, soluble egg antigen from AChE-edited eggs exhibited markedly reduced AChE activity, indicative that programmed Cas9 cleavage mutated the AChE gene. Following injection of AChE-edited schistosome eggs into the tail veins of mice, a significant decrease in circumoval granuloma size was observed in the lungs of the mice. Notably, there was an enhanced Th2 response involving IL-4, −5, −10, and-13 induced by lung cells and splenocytes in mice injected with X5-KI eggs in comparison to control mice injected with unmutated eggs. A Th2-predominant response, with increased levels of IL-4, −13 and GATA3, also was induced by X5 KI eggs in small intestine-draining mesenteric lymph node cells when the gene-edited eggs were introduced into the subserosa of the ileum of the mice. These findings confirmed the potential and the utility of CRISPR/Cas9-mediated genome editing for functional genomics in schistosomes.Author SummarySchistosomiasis is the most devastating of the parasitic helminth diseases. Currently, no vaccines are available for human use and praziquantel is the only available treatment raising considerable concern that drug resistance will develop. A major challenge faced by the schistosomiasis research community is the lack of suitable tools to effectively characterise schistosome gene products as potential new drug and/or vaccine targets. We introduced CRISPR/Cas9 mediated editing into S. mansoni eggs targeting the gene encoding acetylcholinesterase (AChE), a recognized anthelminthic drug target. We found that the major modifications induced by CRISPR/Cas9 in the eggs were generated by homology directed repair (HDR). This platform provides a unique opportunity to generate precise loss-of-function insertions into the schistosome genome. We pre-screened the activity of two guide RNAs of the AChE gene and compared/validated the mutation efficacy using next-generation sequencing analysis at the genomic level and phenotypic modifications at the protein level. That resulted in reduced AChE activity observed in AChE-edited eggs, and decreased lung circumoval granuloma size in mice injected with those edited eggs. The CRISPR/Cas9-genome editing system we established in this study provides a pivotal platform for gene functional studies to identify and test new anti-schistosome intervention targets, which can be extended to the other human schistosome species and other important parasitic helminths.