Max F. Möscheid (Gießen / DE), Prapakorn Wistiphongpun (Washington, DC / US), Wannaporn Ittiprasert (Washington, DC / US), Dr. Thomas Quack (Gießen / DE), Victoria H. Mann (Washington, DC / US), Prof. Dr. Christoph Grunau (Perpignan / FR), Prof. Paul J. Brindley (Washington, DC / US), Prof. Christoph G. Grevelding (Gießen / DE)
Abstract text
"Introduction"
Schistosoma mansoni, a zoonotic parasite, is characterized by a complex lifecycle that includes an invertebrate snail host and a vertebrate final host. Until now, no established protocol exists for the stable transformation of schistosomes.
For investigating the function of a gene of interest (GOI), knock-out models are common for various organisms, but not yet available for trematodes. Until now, RNA interference (RNAi) has been proven as the most suitable method for functional gene characterization. However, RNAi efficiency varies, and it can lead to ectopic effects. CRISPR/Cas-based editing is a powerful tool for gene characterization. To make this technique accessible for trematode research, we tried to establish a protocol for editing a bioinformatically predicted genomic safe harbor site (GSH) of S. mansoni. GSHs are sites in the genome that allow the integration of new genetic material without negatively effecting genome integrity or gene expression.
"Objectives"
The aim of this project was to establish a CRISPR/Cas-based approach to generate transgenic S. mansoni.
"Material and methods"
For editing the identified GSH, a 5'C6-PEG10-modified construct encoding an eGFP reporter-gene driven by a strong native promoter was used as donor DNA. Cas-mediated integration of the transgene was performed by electroporation of eggs. To this end, ribonucleoprotein complexes (RNPs) were used consisting of the enzymes Cas9 or Cas12a and respective guide RNAs targeting the GSH. Integration of the transgene was examined by PCR. Reporter-gene expression was analysed by RT-PCR at the transcript level, and by confocal laser scanning microscopy to reveal eGFP expression.
"Results"
Using both Cas9 and Cas12a, we demonstrated the accessibility of the predicted GSH. Comparison of both Cas-enzymes revealed a significantly higher editing efficiency for Cas12a. Furthermore, we confirmed reporter-gene integration into the selected GSH using both RNPs formed by Cas9 or Cas12a. Finally, eGFP signals were detected demonstrating reporter gene expression in electroporated eggs and miracidia.
"Conclusion"
Successful editing of a predicted schistosomal GSH was shown as well as the expression of a genome-integrated eGFP reporter-gene. These results are proof of concept for a new genome-editing approach in S. mansoni. Our results suggest that both enzymes, Cas9 and Cas12a, are useful for editing the S. mansoni, with a higher efficiency for Cas12a.