Robust and stable transcriptional repression inGiardiausing CRISPRi

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 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.

Length-dependent disassembly maintains four different flagellar lengths in Giardia

“…To assess the contributions of kinesin-13 mediated disassembly to the maintenance of the lengths of all four flagellar pairs, we quantified all flagellar lengths in the CRISPRi-mediated kinesin-13 knockdown (K13kd) strain (Figure 6A, 6B). Using this K13kd strain, we have previously shown that caudal flagellar length increases (McInally et al, 2019). Here we extend this prior work to show that all flagellar pairs have steady-state length increases when kinesin-13 expression is inhibited by ∼60% (Figure 6A, 6B) (McInally et al, 2019), consistent with overexpression of a dominant negative rigor kinesin-13 mutant (Dawson et al, 2007).…”

“…Using this K13kd strain, we have previously shown that caudal flagellar length increases (McInally et al, 2019). Here we extend this prior work to show that all flagellar pairs have steady-state length increases when kinesin-13 expression is inhibited by ∼60% (Figure 6A, 6B) (McInally et al, 2019), consistent with overexpression of a dominant negative rigor kinesin-13 mutant (Dawson et al, 2007). Compared to non-specific gRNA controls, the anterior flagella length in the K13kd strain is increased an average of 1.1±0.04 µm, the posteriolateral by 0.9±0.03 µm, the caudal by 3.1±0.18 µm, and the ventral by 1.6±0.06 µm (Figure 6H).…”

“…Giardia has a single kinesin-13 homolog that localizes to all distal flagellar tips, the median body, and the two mitotic spindles (Dawson et al, 2007). We have shown that kinesin-13 modulates flagellar disassembly in Giardia , as the overexpression of a dominant negative kinesin-13 rigor mutant and the depletion of kinesin-13 by CRISPRi-mediated knockdown cause increased flagellar lengths (Dawson et al, 2007; McInally et al, 2019).…”

“…As in other eukaryotes with IFT-mediated flagellar assembly, conserved IFT components (e.g., IFT trains, BBsome, and kinesin and dynein motors) assemble the membrane-bound regions of each the eight Giardia axonemes (Hoeng et al, 2008; McInally et al, 2019). Cytoplasmic regions of each axoneme may be assembled in an IFT-independent manner as neither kinesin-2 knockdown nor expression of a dominant negative kinesin-2 affect cytoplasmic axoneme length (Dawson et al, 2007; Hoeng et al, 2008).…”

“…This is unlike metazoans, Tetrahymena , and trypanosomes, all of which have multiple kinesin-13 homologs with duplicated or differentiated functions in the various arrays. In Giardia , the ectopic expression of a dominant-negative, kinesin-13 rigor mutant or CRISPRi-mediated knockdown of kinesin-13 (Figure 6) results in dramatically longer flagella (Dawson et al, 2007; McInally et al, 2019). The ultrastructure of the all eight axonemes retain the canonical 9+2 arrangement of doublet microtubules but lack a flagellar tip complex (Dawson et al, 2007).…”