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Abstract:
Studies of gene function within the important model parasitic species Strongyloides ratti have long been stymied by a lack of appropriate genetic tools. While CRISPR/Cas9 has recently been successfully reported for S. stercoralis, it did not work as effectively in S. ratti. To counter this we recently succeeded in establishing RNAi in S. ratti. By firstly determining if S. ratti had the requisite proteins within its genome, we then developed a protocol for RNAi by soaking, by systematically varying the soaking medium, duration of soaking, addition of pharyngeal-pump- inducing compounds, the age of larvae and the soaking temperature. By using siRNAs, we bypass the need for dsRNA processing which Strongyloides appears to be refractory to. This approach was successful, with three separate genes so far knocked down with minimal off- target effects detected. This approach also has the added benefit of allowing analysis of gene function at different stages of the life cycle that occur outside a host, without the need of host passage, which means we do not have to maintain multiple mutant lines in a substantial amount of laboratory animals which is both laborious and expensive. We then used this technique to study the important gene daf-12, which has long been proposed to have role in infective larvae development within Strongyloides based upon the external application of dafachronic acid and gene expression studies. We found that suppression of daf-12 severely impairs the formation of infective larvae and redirects development towards free-living larvae. We also found that daf- 12 functions in other processes (e.g. fat metabolism or temperature stress tolerance) are conserved in S. ratti, compared with C. elegans. Our experiments also confirm that the previously reported pharmacological effects of dafachronic acid within Strongyloides acted through DAF-12. Inhibitory molecules with optimized pharmacological properties can now be developed based on the DAF-12 structure.
