A web portal for gene expression across all life stages of <i>Schistosoma mansoni</i>

Lu, Zhongqiu; Zhang, Y; Berriman, Matt

doi:10.1101/308213

Cited by 25 publications

(49 citation statements)

References 6 publications

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“…The biological function of SULT-OR remains unknown, except that it converts the pro-drugs OXA and hycanthone to their active forms [23,24]. It displays sulfotransferase activity in vitro on exogenous substrates [23], even though the protein shows a low level of sequence similarity to other sulfotransferases, and it is mostly expressed in the intra-mammalian stages of the life cycle (schistosomula and adults, Supplementary Figure S2A) [25]. Intriguingly, SULT-OR belongs to a gene family that has expanded in trematodes [37], suggesting it may play an important role in clade-specific biology.…”

Section: The Sult-or Gene Belongs To a Multi-copy Locus On Chromosomementioning

confidence: 99%

“…We chose the SULT-OR sulfotransferase (Smp_089320) gene as a target because recessive mutations in this gene, both induced in laboratory conditions and detected in field samples, confer resistance to the drug oxamniquine (OXA) [23,24]. In addition, it is mostly expressed in the intra-mammalian stages of the life cycle (schistosomula and adults) [25] that would likely be the target of any new intervention strategy. Our findings provide insights that will help pave the way towards using CRISPR-Cas to achieve the generation of stable genetically-engineered schistosomes.…”

Section: Introductionmentioning

confidence: 99%

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

Sankaranarayanan

Coghlan

Driguez

et al. 2020

Preprint

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At least 250 million people worldwide suffer from schistosomiasis, caused by Schistosoma worms. Genome sequences for several Schistosoma species are available, including a highquality 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. Here, 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. 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. 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.

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Section: The Sult-or Gene Belongs To a Multi-copy Locus On Chromosomementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Sankaranarayanan

Coghlan

Driguez

et al. 2020

Preprint

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“…This layer is metabolically active (27) and is considered to be the site of synthesis of the ω1 T2 ribonuclease that is released from the egg into the granuloma (25, 27, 29), along with other secreted/ excreted proteins that facilitate egress of the egg from the blood vessels and through the wall of the intestine (27). Expression of ω1 is developmentally tightly regulated: based on comparison of expression in the egg to expression in the miracidium, immunostaining and transmission electron microscopy of the mature egg, and on meta-analysis of transcriptomic findings, all or most expression of ω1 occurs solely in the mature egg; expression does not occur in the immature egg or other developmental stages (20, 25, 27, 29, 72, 73) (Fig. s1c).…”

Section: Discussionmentioning

confidence: 99%

“…An alternative explanation for the paradox may be the tight tissue specific expression of ω1, which not only is expressed only in the egg developmental stage of S. mansoni (Fig. S1C) [70] and, moreover, only occurs within the syncytial ‘inner envelope’ of squamous, epidermal-like cells surrounding the miracidium [66]. Expression of ω1 appears to be tightly developmentally regulated [35]; based on comparison of expression in the egg to expression in the miracidium, immunostaining and transmission electron microscopy of the mature egg, all or most expression of ω1 takes place in the mature egg and not in immature eggs or other larval or adult stages [20, 25, 27, 29, 71].…”

Section: Discussionmentioning

confidence: 99%

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

Ittiprasert

Mann

Karinshak

et al. 2018

Preprint

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34 35 CRISPR/Cas9 based genome editing has not yet been reported in schistosomes. Here, we tested 36 this approach by targeting omega-1 (ω1) of Schistosoma mansoni as a proof of principle. This 37 secreted ribonuclease is crucial for Th2 priming and granuloma formation, providing informative 38 immuno-pathological readouts for programmed genome editing. Schistosome eggs were either 39 exposed to recombinant Cas9 complexed with a synthetic guide RNA (sgRNA) complementary 40 to exon 6 of ω1 by electroporation or transduced with pseudotyped lentivirus encoding Cas9 and 41 the sgRNA. Some eggs were also transduced with a single stranded oligodeoxynucleotide donor 42 transgene that encoded six stop codons, flanked by 50 nt-long 5'-and 3'-microhomology arms 43 matching the predicted Cas9-catalyzed double stranded break (DSB) within ω1. CRISPResso 44 analysis of amplicons spanning the DSB revealed ~4.5% of the reads were mutated by insertions, 45deletions and/or substitutions, with an efficiency for homology directed repair of 0.19% insertion 46 of the donor transgene. Transcripts encoding ω1 were reduced >80%, and lysates of ω1-edited 47 eggs displayed diminished ribonuclease activity indicative that programmed editing mutated the 48 ω1 gene. Whereas soluble lysates of wild type eggs polarized Th2 cytokine responses including 49 IL-4 and IL-5 in human macrophage/T cell co-cultures, diminished levels of these cytokines 50followed the exposure to those of ω1-mutated schistosome eggs. Following injection of 51 schistosome eggs into the tail vein of BALB/c mice, the volume of pulmonary granulomas 52surrounding ω1-mutated eggs was 18-fold smaller than wild type eggs. Programmed genome 53 editing was active in schistosomes, Cas9-catalyzed chromosomal breakage was repaired by 54 homology directed repair and/or non-homologous end joining, and mutation of ω1 impeded the 55 capacity of schistosome eggs both to drive Th2 polarization and to provoke formation of 56 pulmonary circumoval granulomas. Knock-out of ω1 and the impaired immunological phenotype 57showcase the novel application of programmed gene editing in and functional genomics for 58 schistosomes. 59 60 61 Introduction 62 63Schistosomiasis is considered the most problematic of the human helminth diseases in terms of 64 morbidity and mortality [1][2][3][4]. The past decade has seen major advances in knowledge and 65 understanding of the pathophysiology, developmental biology, evolutionary relationships and 66 genome annotation of the human schistosomes [5][6][7][8][9][10][11][12][13][14][15][16]. Establishing CRISPR/Cas9 genome 67 editing in schistosomiasis would greatly enable effective functional genomics approaches. The 68 stable CRISPR/Cas9-based site-specific gene mutation and phenotyping will drive innovation 69 and a deeper understanding of schistosome pathogenesis, biology and evolution [17]. 70 71The schistosome egg plays a central role both in disease transmission and pathogenesis [1]. The 72 appearance of S. mansoni eggs in host tissues by six to seven week...

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Small Molecule Ligands of the BET-like Bromodomain, SmBRD3, Affect Schistosoma mansoni Survival, Oviposition, and Development

Schiedel,

McArdle,

Padalino

et al. 2023

J. Med. Chem.

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Schistosomiasis is a disease affecting >200 million people worldwide, but its treatment relies on a single agent, praziquantel. To investigate new avenues for schistosomiasis control, we have conducted the first systematic analysis of bromodomain-containing proteins (BCPs) in a causative species, Schistosoma mansoni . Having identified 29 putative bromodomains (BRDs) in 22 S. mansoni proteins, we selected Sm BRD3, a tandem BRD-containing BCP that shows high similarity to the human bromodomain and extra terminal domain (BET) family, for further studies. Screening 697 small molecules identified the human BET BRD inhibitor I-BET726 as a ligand for Sm BRD3. An X-ray crystal structure of I-BET726 bound to the second BRD of Sm BRD3 [ Sm BRD3(2)] enabled rational design of a quinoline-based ligand ( 15 ) with an ITC K d = 364 ± 26.3 nM for Sm BRD3(2). The ethyl ester pro-drug of compound 15 (compound 22 ) shows substantial effects on sexually immature larval schistosomula, sexually mature adult worms, and snail-infective miracidia in ex vivo assays.

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A web portal for gene expression across all life stages of Schistosoma mansoni

Cited by 25 publications

References 6 publications

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

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

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

Small Molecule Ligands of the BET-like Bromodomain, SmBRD3, Affect Schistosoma mansoni Survival, Oviposition, and Development

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