Abstract Giardia lamblia is a binucleate protistan parasite causing significant diarrheal disease worldwide. An inability to target Cas9 to both nuclei, combined with the lack of non-homologous end joining and markers for positive selection, has stalled the adaptation of CRISPR/Cas9-mediated genetic tools for this widespread parasite. CRISPR interference (CRISPRi) is a modification of the CRISPR/Cas9 system that directs catalytically inactive Cas9 (dCas9) to target loci for stable transcriptional repression. Using a Giardia nuclear localization signal to target dCas9 to both nuclei, we developed efficient and stable CRISPRi-mediated transcriptional repression of exogenous and endogenous genes in Giardia . Specifically, CRISPRi knockdown of kinesin-2a and kinesin-13 causes severe flagellar length defects that mirror defects with morpholino knockdown. Knockdown of the ventral disc MBP protein also causes severe structural defects that are highly prevalent and persist in the population more than five days longer than transient morpholino-based knockdown. By expressing two gRNAs in tandem to simultaneously knock down kinesin-13 and MBP, we created a stable dual knockdown strain with both flagellar length and disc defects. The efficiency and simplicity of CRISPRi in polyploid Giardia allows for rapid evaluation of knockdown phenotypes and highlights the utility of CRISPRi for emerging model systems.

Robust and stable transcriptional repression inGiardiausing CRISPRi