Department of Parasitology, College of Medicine, National Taiwan University, Taipei, Taiwan, ROC.
PLoS One. 2019 Mar 11;14(3):e0213594. doi: 10.1371/journal.pone.0213594. eCollection 2019.
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.
蓝氏贾第鞭毛虫通过分化为耐水的囊来进入休眠状态，这种囊可以感染新的宿主。这种分化的基本特征是合成三种囊壁蛋白（CWPs）。髓样白血病因子（MLF）蛋白参与哺乳动物的细胞分化和肿瘤发生，但对其在原生动物寄生虫中的作用知之甚少。我们开发了 CRISPR/Cas9 系统来了解 MLF 在贾第虫中的作用。由于贾第虫的两个核中有四倍体基因组，因此很难完全破坏贾第虫中的一个基因。我们仅产生了敲低而不是敲除突变体。我们发现，mlf 基因的敲低导致 cwp 基因表达和囊形成显著减少，表明 MLF 在囊形成中起积极作用。我们进一步将 mlf 用作模型基因来改进该系统。添加 NHEJ 抑制剂 Scr7，或将所有 gRNA 和 Cas9 表达盒组合到一个质粒中，可提高基因敲除效率，并显著降低 cwp 基因表达。我们的结果提供了关于 MLF 在诱导贾第虫分化中的积极作用的见解，并为贾第虫研究提供了有用的工具。
