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 nonhomologous 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 disk MBP protein also causes severe structural defects that are highly prevalent and persist in the population more than 5 d longer than defects associated with transient morpholino-based knockdown. By expressing two guide RNAs in tandem to simultaneously knock down kinesin-13 and MBP, we created a stable dual knockdown strain with both flagellar length and disk defects. The efficiency and simplicity of CRISPRi in polyploid Giardia allows rapid evaluation of knockdown phenotypes and highlights the utility of CRISPRi for emerging model systems.

Giardia is a single-celled parasite causing significant diarrheal disease in several hundred million people worldwide. Due to limited access to the site of infection in the gastrointestinal tract, our understanding of the dynamics of Giardia infections in the host has remained limited and largely inferred from laboratory culture. To better understand Giardia physiology and colonization in the host, we developed imaging methods to quantify Giardia expressing bioluminescent physiological reporters in two relevant animal models. We discovered that parasites primarily colonize and encyst in the proximal small intestine in discrete, high-density foci. We also show that high parasite density contributes to encystation initiation.

With eight flagella of four different lengths, the parasitic protist Giardia is an ideal model to evaluate flagellar assembly and length regulation. To determine how four different flagellar lengths are maintained, we used live-cell quantitative imaging and mathematical modeling of conserved components of intraflagellar transport (IFT)-mediated assembly and kinesin-13-mediated disassembly in different flagellar pairs. Each axoneme has a long cytoplasmic region extending from the basal body, and transitions to a canonical membrane-bound flagellum at the ‘flagellar pore’. We determined that each flagellar pore is the site of IFT accumulation and injection, defining a diffusion barrier functionally analogous to the transition zone. IFT-mediated assembly is length-independent, as train size, speed, and injection frequencies are similar for all flagella. We demonstrate that kinesin-13 localization to the flagellar tips is inversely correlated to flagellar length. Therefore, we propose a model where a length-dependent disassembly mechanism controls multiple flagellar lengths within the same cell.

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Giardia is a highly prevalent, yet understudied protistan parasite causing diarrheal disease worldwide. Hosts ingest Giardia cysts from contaminated sources. In the gastrointestinal tract, cysts excyst to become motile trophozoites, colonizing and attaching to the gut epithelium. Trophozoites later differentiate into infectious cysts that are excreted and contaminate the environment. Due to the limited accessibility of the gut, the temporospatial dynamics of giardiasis in the host is largely inferred from laboratory culture and thus may not mirror Giardia physiology in the host. Here we have developed bioluminescent imaging (BLI) to directly interrogate and quantify the in vivo temporospatial dynamics of giardiasis, thereby providing an improved murine model to evaluate anti-Giardia drugs. Using BLI, we determined that parasites primarily colonize the proximal small intestine non-uniformly in high-density foci. By imaging encystation-specific bioreporters, we show that encystation initiates shortly after inoculation and continues throughout the entire duration of infection. Encystation also initiates in high-density foci in the proximal small intestine, and high-density laboratory cultures of parasites are also stimulated to encyst. This work overturns the assumption that parasites encyst later during infection as they are dislodged and travel through the colon. We suggest that these high-density regions of parasite colonization likely result in localized pathology to the epithelium, and encystation occurs when trophozoites reach a threshold density due to local nutrient depletion. This more accurate visualization of giardiasis redefines the dynamics of in vivo Giardia life cycle, paving the way for future mechanistic studies of density-dependent parasitic processes in the host.SignificanceGiardia is a single-celled parasite causing both acute and chronic diarrheal disease in over one billion people worldwide. Due to limited access to the site of infection in the gastrointestinal tract, our understanding of the dynamics of Giardia infections in the host has remained limited, and largely inferred from laboratory culture. To better understand giardiasis in the host, we developed imaging methods to quantify Giardia expressing bioluminescent physiological reporters in live mice. We discovered that parasites primarily colonize and encyst in the proximal small intestine in discrete, high-density foci. Furthermore, this work provides evidence of a parasite density-based threshold for the differentiation of Giardia into cysts in the host. These findings overturn existing paradigms of giardiasis infection dynamics in the host.