Gene knockdown in malaria parasites via non-canonical RNAi. Plasmodium berghei

To introduce a non-canonical RNA interference pathway into Plasmodium berghei (P. berghei), we generated a P. berghei line constitutively expressing the human protein Argonaute 2 (Ago2) and episomally expressed Ago2-dependent short hairpin RNAs (AgoshRNAs) against GFP. Using RNA sequencing (RNA-Seq), we analyzed transcriptomes of wild type parasites (PbGFPcon), Ago2-expressing parasites (PbAgo2) and PbAgo2 parasites expressing a scrambled AgoshRNA (scr) or anti-GFP-AgoshRNA (aGFP). We compared PbAgo2 to wildtype PbGFPcon parasites and find that Ago2 expression interferes with the expression of development-of-zygote-inhibited (DOZI)-dependent transcripts. When comparing PbAgo2 plus scr with PbAgo2 expressing aGFP, we find a clear knockdown of GFP; Other than that, we do not observe a significant off-targeting in these samples besides differential expression of genes belonging to multi-gene families. Overall design: For PbGFPcon, two independently prepared technical replicates were analyzed. For all other samples (PbAgo2, PbAgo2 + scr, PbAgo2 + aGFP) two biological replicates resulting from independent transfections were analyzed.