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

Abstract

Background. At least 250 million people worldwide suffer from schistosomiasis, caused by Schistosoma worms. Genome sequences for several Schistosoma species are available, including a high-quality 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. Methods. 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. Results. 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. Conclusions. 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.

CRISPR/Cas9: A new tool for the study and control of helminth parasites

“…achieved CRISPR/Cas9‐based knockout in S. mansoni targeting the SULT‐OR gene in which mutations confer resistance to the drug oxamniquine. [ 49 ] Using electroporation to deliver in vitro assembled ribonucleoprotein (RNP) complexes by combining specific sgRNA with the Cas9 nuclease to adult worms, mother sporocysts and eggs, variable gene mutation efficiency was evident across the different life cycle stages. [ 49 ] The highest SULT‐OR modification efficiency was observed in adult worms (0.3%‐2% deletions), followed by sporocysts (0.1%‐0.2% deletions) but no NHEJ‐mediated modifications were detected in eggs, [ 49 ] observations supporting those of You et al.…”

“…Possible explanations for the observed differences in gene mutation efficiency can be summarized as follows: (1) Adult worms have a larger surface area to volume ratio than sporocysts and eggs, a feature that may more readily facilitate the delivery of CRISPR components by electroporation. [ 49 ] (2) There are higher expression levels of some key enzymes in the NHEJ pathway in adult worms than in sporocysts and eggs, an observation that suggests that DNA repair activities in the NHEJ pathway may be more efficient in adults. [ 49 ] (3) The egg shell of S. mansoni has three layers and is a hardened and tanned structure.…”

“…To date, the CRISPR/Cas9 gene editing system has been successfully established in four free‐living nematodes ( Caenorhabditis elegans , [ 17,35–42 ] Pristionchus pacificus , [ 21,43 ] Auanema rhodensis and A. freiburgensis [ 44 ] ), three parasitic nematodes (two human parasites, Strongyloides stercoralis and Brugia malayi , and a parasite of rats, Strongyloides ratti [ 20,44–47 ] ), and two parasitic trematodes (the human liver fluke Opisthorchis viverrini and the human blood fluke Schistosoma mansoni [ 22,23,48,49 ] ). In these studies, Cas9 and target specific sgRNAs were expressed using either an in vivo plasmid‐based system or by in vitro synthesis of the CRISPR components.…”

Genetic architecture of transmission stage production and virulence in schistosome parasites

“…We have tried several approaches to induce RNAi in daughter sporocysts including injection of ds or siRNA into the snail hemolymph/tissues, but have so far not been able to knockdown genes in this stage. CRISPR provide a possible alternative approach but is still at an early stage of development for S. mansoni (Ittiprasert et al, 2019;Sankaranarayanan et al, 2020;You et al, 2021). Once effective gene manipulation approaches are available for late sporocysts we will be able to directly examine the involvement of our leading candidate genes.…”

Urogenital Schistosomiasis—History, Pathogenesis, and Bladder Cancer

“…In 2009 schistosome species moved into the post-genomic era, as the first draft genomes for S. mansoni and S. japonicum were published [ 32 , 33 ], and a few years later the S. haematobium genome was released [ 34 ]. In the last decade, a remarkable functional technology development has occurred, from culture system refinements [ 35 , 36 ], the use of functional tools, such as transgenesis and genome editing [ 37 , 38 , 39 , 40 ], to single cell transcriptomics [ 41 ], has accelerated the discovery of targets for novel control strategies…”