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Giardia lamblia  becomes dormant by differentiation into a water-resistant cyst that can infect a new host. Synthesis of three cyst wall proteins (CWPs) is the fundamental feature of this differentiation. Myeloid leukemia factor (MLF) proteins are involved in cell differentiation, and tumorigenesis in mammals, but little is known about its role in protozoan parasites. We developed a CRISPR/Cas9 system to understand the role of MLF in  Giardia . Due to the tetraploid genome in two nuclei of  Giardia , it could be hard to disrupt a gene completely in  Giardia . We only generated knockdown but not knockout mutants. We found that knockdown of the  mlf  gene resulted in a significant decrease of  cwp  gene expression and cyst formation, suggesting a positive role of MLF in encystation. We further used  mlf  as a model gene to improve the system. The addition of an inhibitor for NHEJ, Scr7, or combining all cassettes for gRNA and Cas9 expression into one plasmid resulted in improved gene disruption efficiencies and a significant decrease in  cwp  gene expression. Our results provide insights into a positive role of MLF in inducing  Giardia  differentiation and a useful tool for studies in  Giardia .

Development of CRISPR/Cas9-mediated gene disruption systems in Giardia lamblia