Targeted mutagenesis in a human-parasitic nematode

Gang, Spencer S.; Castelletto, Michelle L.; Bryant, Astra S.; Yang, Emily; Mancuso, Nicholas; Lopez, Jacqueline B.; Pellegrini, Matteo; Hallem, Elissa A.

doi:10.1371/journal.ppat.1006675

Cited by 112 publications

(219 citation statements)

References 56 publications

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“…Large CRISPR-induced deletions of >500 bp have been observed in the nematode Strongyloides stercoralis [20]. In addition to the most common CRISPR-Cas9-induced deletions observed in S. mansoni that extended 34 bp upstream of the predicted Cas9 cut site ( Figure 3B), we did observe low-frequency deletions (supported by few reads) extending from the predicted Cas9 cut site to 102 bp upstream (to position 36 in the reference amplicon) (Figure 4 and Supplementary Figure S8).…”

Section: Evidence For Large Deletionsmentioning

confidence: 99%

“…Site-specific integration of transgenes and highly precise site-specific genome editing using CRISPR-Cas technology will be a key step [19]. 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].…”

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: Evidence For Large Deletionsmentioning

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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“…There is growing evidence that chemosensory and other sensory modalities play an important role in nematode parasite transmission and intra-host migration (11)(12)(13)(14)(15)(16)(17)(18)(19)(20) . However, most studies have focused on single-host nematode parasites with direct life cycles, which are phylogenetically distant from the vector-borne filarial parasites of clade III.…”

Section: Introductionmentioning

confidence: 99%

Genetic and functional diversification of chemosensory pathway receptors in mosquito-borne filarial nematodes

Wheeler

Heimark

Airs

et al. 2019

Preprint

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Lymphatic filariasis (LF) afflicts over 60 million people worldwide and leads to severe pathological outcomes in chronic cases. The filarial nematode parasites that cause LF require both arthropod (mosquito) intermediate hosts and mammalian definitive hosts for their propagation. The invasion and migration of filarial parasites through host tissues are complex and critical to survival, yet little is known about the receptors and signaling pathways that mediate taxis in these medically important species.To better understand filarial chemosensation we employ comparative genomics, transcriptomics, reverse genetics, and chemical approaches to identify putative chemosensory receptor proteins and perturb chemosensory phenotypes in filarial nematode parasites. We find that chemoreceptor family size is correlated with the presence of environmental (extra-host) stages in nematode life cycles, and that filarial parasites contain a compact and highly-diverged chemoreceptor complement and lineage-specific ion channels that are predicted to operate downstream of chemoreceptor activation. In Brugia malayi, an etiological agent of LF, chemoreceptor expression patterns correspond to distinct parasite migration events across the life cycle. To interrogate the role of chemosensation in host migration, arthropod infectious stage (microfilariae) and vertebrate infectious stage (L3) Brugia parasites were incubated in nicotinamide, an agonist of the nematode transient receptor potential (TRP) channel osm-9 . Exposure of microfilariae to nicotinamide alters intra-mosquito migration while exposure of L3s reduces chemotaxis towards host-associated cues in vitro . Nicotinamide exposure also modulates thermosensory responses in L3s, suggesting a polymodal sensory role for Brugia osm-9 . Reverse genetic studies implicate both osm-9 and the cyclic nucleotide-gated (CNG) channel subunit tax-4 in larval chemotaxis towards host serum, while these ion channel subunits do not rescue chemosensory defects in C. elegans osm-9 and tax-4 knock-out strains. Together, these data reveal genetic and functional diversification of chemosensory signaling proteins in filarial nematode parasites, and encourage a more thorough investigation of clade and parasite-specific facets of nematode sensory receptor biology.

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“…Large DNA deletions have been associated with CRISPR/Cas9 mutations in another helminth species (42). However, using qPCR to estimate relative copy number, as previously described (49), evidence that silencing of ω1 was associated with a reduction in the copy number of this multi-gene locus was not seen (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Notably, less than 5% efficiency in gene editing, based on analysis of the NGS reads, appeared to account for this markedly reduced (>80%) gene expression. The possibility of large-scale deletions, as reported in Strongyloides stercoralis [40], offered one explanation for this apparent paradox. However, qPCR analysis of ω1 copy number did not reveal apparent differences between treatments and controls.…”

Section: Discussionmentioning

confidence: 99%

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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Targeted mutagenesis in a human-parasitic nematode

Cited by 112 publications

References 56 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

Genetic and functional diversification of chemosensory pathway receptors in mosquito-borne filarial nematodes

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

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