Gene knockdown in malaria parasites via non-canonical RNAi

Nucleic Acids Res. 2020 Jan 10;48(1):e2. doi: 10.1093/nar/gkz927.


The lack of endogenous RNAi machinery in the malaria parasite Plasmodium hampers gene annotation and hence antimalarial drug and vaccine development. Here, we engineered rodent Plasmodium berghei to express a minimal, non-canonical RNAi machinery that solely requires Argonaute 2 (Ago2) and a modified short hairpin RNA, so-called AgoshRNA. Using this strategy, we achieved robust and specific gene knockdown throughout the entire parasite life cycle. We also successfully silenced the endogenous gene perforin-like protein 2, phenocopying a full gene knockout. Transcriptionally restricting Ago2 expression to the liver stage further enabled us to perform a stage-specific gene knockout. The RNAi-competent Plasmodium lines reported here will be a valuable resource for loss-of-function phenotyping of the many uncharacterized genes of Plasmodium in low or high throughput, without the need to engineer the target gene locus. Thereby, our new strategy and transgenic Plasmodium lines will ultimately benefit the discovery of urgently needed antimalarial drug and vaccine candidates. Generally, the ability to render RNAi-negative organisms RNAi-competent by mere introduction of two components, Ago2 and AgoshRNA, is a unique paradigm that should find broad applicability in other species.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Anopheles / parasitology
  • Argonaute Proteins / genetics*
  • Argonaute Proteins / metabolism
  • Female
  • Genes, Reporter
  • Genetic Engineering / methods*
  • Green Fluorescent Proteins / antagonists & inhibitors
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Life Cycle Stages / genetics
  • Mice
  • Mice, Inbred C57BL
  • Mosquito Vectors / parasitology
  • Organisms, Genetically Modified
  • Perforin / genetics
  • Perforin / metabolism
  • Plasmodium berghei / genetics*
  • Plasmodium berghei / growth & development
  • Plasmodium berghei / metabolism
  • Protozoan Proteins / antagonists & inhibitors
  • Protozoan Proteins / genetics*
  • Protozoan Proteins / metabolism
  • RNA Interference*
  • RNA, Small Interfering / genetics*
  • RNA, Small Interfering / metabolism
  • Transgenes


  • Argonaute Proteins
  • Protozoan Proteins
  • RNA, Small Interfering
  • Perforin
  • Green Fluorescent Proteins