Robust and stable transcriptional repression in Giardia using CRISPRi

Area of Science:

• Microbiology
• Parasitology
• Molecular Biology

Background:

• Giardia lamblia is a significant global cause of diarrheal disease.
• Existing CRISPR/Cas9 tools are difficult to adapt for Giardia due to its unique biology (binucleate, lack of NHEJ).
• CRISPR interference (CRISPRi) offers a stable gene repression method using catalytically inactive Cas9 (dCas9).

Purpose of the Study:

• To develop and validate CRISPRi for efficient and stable gene knockdown in Giardia lamblia.
• To investigate the function of specific genes (kinesin-2a, kinesin-13, MBP) by observing knockdown phenotypes.
• To establish a dual knockdown system for studying complex genetic interactions in Giardia.

Main Methods:

• Engineered dCas9 with a Giardia nuclear localization signal to target both nuclei.
• Designed guide RNAs for CRISPRi-mediated transcriptional repression of exogenous and endogenous genes.
• Created a dual knockdown strain using tandem guide RNAs to target multiple genes simultaneously.

Main Results:

• Achieved efficient and stable CRISPRi-mediated gene repression in Giardia.
• Demonstrated severe flagellar length defects upon knockdown of kinesin-2a and kinesin-13.
• Observed significant structural defects in the ventral disk after knockdown of the MBP protein, with prolonged persistence.
• Successfully generated a stable dual knockdown strain exhibiting both flagellar and ventral disk defects.

Conclusions:

• CRISPRi is an effective tool for stable gene knockdown in the polyploid parasite Giardia lamblia.
• This system allows for rapid evaluation of gene function and knockdown phenotypes.
• CRISPRi represents a valuable genetic tool for Giardia research and other emerging model systems.