Dataset Information

Targeted insertion and reporter transgene activity at a gene safe harbor of the human blood fluke, Schistosoma mansoni.

ABSTRACT: The identification and characterization of genomic safe harbor sites (GSHs) can facilitate consistent transgene activity with minimal disruption to the host cell genome. We combined computational genome annotation and chromatin structure analysis to predict the location of four GSHs in the human blood fluke, Schistosoma mansoni, a major infectious pathogen of the tropics. A transgene was introduced via CRISPR-Cas-assisted homology-directed repair into one of the GSHs in the egg of the parasite. Gene editing efficiencies of 24% and transgene-encoded fluorescence of 75% of gene-edited schistosome eggs were observed. The approach advances functional genomics for schistosomes by providing a tractable path for generating transgenics using homology-directed, repair-catalyzed transgene insertion. We also suggest that this work will serve as a roadmap for the development of similar approaches in helminths more broadly.

SUBMITTER: Ittiprasert W

PROVIDER: S-EPMC10391569 | biostudies-literature | 2023 Jul

REPOSITORIES: biostudies-literature

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Publications

Targeted insertion and reporter transgene activity at a gene safe harbor of the human blood fluke, <i>Schistosoma mansoni</i>.

Ittiprasert Wannaporn W Moescheid Max F MF Chaparro Cristian C Mann Victoria H VH Quack Thomas T Rodpai Rutchanee R Miller André A Wisitpongpun Prapakorn P Buakaew Watunyoo W Mentink-Kane Margaret M Schmid Sarah S Popratiloff Anastas A Grevelding Christoph G CG Grunau Christoph C Brindley Paul J PJ

Cell reports methods 20230724 7

The identification and characterization of genomic safe harbor sites (GSHs) can facilitate consistent transgene activity with minimal disruption to the host cell genome. We combined computational genome annotation and chromatin structure analysis to predict the location of four GSHs in the human blood fluke, <i>Schistosoma mansoni</i>, a major infectious pathogen of the tropics. A transgene was introduced via CRISPR-Cas-assisted homology-directed repair into one of the GSHs in the egg of the par ...[more]

PMID: 37533651

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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

Targeted insertion and reporter transgene activity at a gene safe harbor of the human blood fluke, Schistosoma mansoni.

Publications

Targeted insertion and reporter transgene activity at a gene safe harbor of the human blood fluke, <i>Schistosoma mansoni</i>.

Similar Datasets

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