Schistosoma mansoni: Off-target analyses using nonspecific double-stranded RNAs as control for RNAi experiments in schistosomula

Abstract

RNA interference is a well established and widely used reverse genetic tool available for gene functional studies in trematodes. This technique requires the use of nonrelevant double-stranded RNA as control. However, several authors have reported inconsistencies associated with RNAi. We used RNASeq to analyze genes affected by nonspecific dsRNA exposure. We found only few genes presenting altered expression in schistosomula exposed to GFP or mCherry nonspecific-dsRNAs, most of them encoding uncharacterized proteins. Correlation analysis revealed that there are more differences among biological replicates, than due to treatment with nonspecific controls. These observations are of key relevance to other RNAi gene function assessment in other organisms.

A metalloprotease produced by larval Schistosoma mansoni facilitates infection establishment and maintenance in the snail host by interfering with immune cell function

“…While snails infected with GFP KD miracidia still demonstrated a rise in SmLeish transcription levels peaking at four days post infection, those infected with SmLeish KD miracidia featured no such increase, confirming specific targeting of the SmLeish mRNA. While this does not control for the possibility of off-target impacts of the administration of the siRNA oligos, the inclusion of the GFP-specific siRNA control accounts for the possibility that any off-target effects might impact the outcome of the encapsulation or chemokinesis bioassays [ 57 ]. Protein expression was also affected by the siRNA treatment, with the soluble ~48 kDa variant becoming undetectable in whole snail lysates, while the ~130 kDa variant exhibited a decrease in expression at 4 days post infection.…”

Disminución del impacto ambiental en el control del parásito Haemonchus contortus en ovinos

“…El silenciamiento de genes es un mecanismo de conservación que su función está dada por proteger las células a través del ARN de (Khalil, Foda, Suresh, & Singh, 2016), Schistosoma mansoni (Gava et al, 2017),…”

Defining an optimal control for RNAi experiments with adult Schistosoma mansoni

“…However, are these non-homologous sequences as suitable as commonly assumed against the background that they are chopped into 19–23 nt long siRNA pieces, which may accidentally find unwanted target sequences? The possibility that dsRNAs can exert off-target effects has been demonstrated in different biological systems 14 (like C. elegans 15 , mammalian cells 16 and D. melanogaster 17 ), including schistosomes and other parasites ( S. mansoni 18 , S. japonicum 19 , Ascaris suum 20 and Fasciola gigantia 21 ). In addition, treatment with high concentrations of dsRNA poses the danger of saturation of the RISC complex, which might lead to systemic dysregulation of gene expression 22 – 24 .…”