Dataset Information

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.

SUBMITTER: Ittiprasert W

PROVIDER: S-EPMC6355194 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Publications

Programmed genome editing of the omega-1 ribonuclease of the blood fluke, <i>Schistosoma mansoni</i>.

Ittiprasert Wannaporn W Mann Victoria H VH Karinshak Shannon E SE Coghlan Avril A Rinaldi Gabriel G Sankaranarayanan Geetha G Chaidee Apisit A Tanno Toshihiko T Kumkhaek Chutima C Prangtaworn Pannathee P Mentink-Kane Margaret M MM Cochran Christina J CJ Driguez Patrick P Holroyd Nancy N Tracey Alan A Rodpai Rutchanee R Everts Bart B Hokke Cornelis H CH Hoffmann Karl F KF Berriman Matthew M Brindley Paul J PJ

eLife 20190115

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 <i>Schistosoma mansoni</i> 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. Seq ...[more]

PMID: 30644357

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Project description:Blood flukes of the genus Schistosoma are platyhelminth parasites that infect 200 million people worldwide. Digestion of nutrients from the host bloodstream is essential for parasite development and reproduction. A network of proteolytic enzymes (proteases) facilitates hydrolysis of host hemoglobin and serum proteins.We identified a new cathepsin L termed SmCL3 using PCR strategies based on S. mansoni EST sequence data. An ortholog is present in Schistosoma japonicum. SmCL3 was heterologously expressed as an active enzyme in the yeast, Pichia pastoris. Recombinant SmCL3 has a broad pH activity range against peptidyl substrates and is inhibited by Clan CA protease inhibitors. Consistent with a function in degrading host proteins, SmCL3 hydrolyzes serum albumin and hemoglobin, is localized to the adult gastrodermis, and is expressed mainly in those life stages infecting the mammalian host. The predominant form of SmCL3 in the parasite exists as a zymogen, which is unusual for proteases. This zymogen includes an unusually long prodomain with alpha helical secondary structure motifs. The striking specificity of SmCL3 for amino acids with large aromatic side chains (Trp and Tyr) at the P2 substrate position, as determined with positional scanning-synthetic combinatorial library, is consistent with a molecular model that shows a large and deep S2 pocket. A sequence similarity network (SSN) view clusters SmCL3 and other cathepsins L in accordance with previous large-scale phylogenetic analyses that identify six super kingdoms.SmCL3 is a gut-associated cathepsin L that may contribute to the network of proteases involved in degrading host blood proteins as nutrients. Furthermore, this enzyme exhibits some unusual sequence and biophysical features that may result in additional functions. The visualization of network inter-relationships among cathepsins L suggests that these enzymes are suitable 'marker sequences' for inclusion in future phylogenetic analyses.

Dataset Information

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

Publications

Programmed genome editing of the omega-1 ribonuclease of the blood fluke, <i>Schistosoma mansoni</i>.

Similar Datasets

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