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

PLoS One. 2019 Mar 11;14(3):e0213594. doi: 10.1371/journal.pone.0213594. eCollection 2019.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CRISPR-Cas Systems*
  • Gene Expression Regulation
  • Giardia lamblia / cytology
  • Giardia lamblia / genetics*
  • Giardiasis / parasitology
  • Humans
  • Plasmids / genetics
  • Protozoan Proteins / genetics*

Substances

  • Protozoan Proteins

Grants and funding

This work was supported by the Ministry of Science and Technology (grant numbers MOST 99-2320-B-002-017-MY3, 100-2325-B-002-039, 101-2325-B-002-036-, 103-2628-B-002-006-MY3-, and 106-2320-B-002 -038 -MY2, PI: CH Sun); and the National Health Research Institutes (grant number NHRI-EX99-9510NC, PI: CH Sun) in Taiwan, and was also supported in part by the Department of Medical Research in National Taiwan University Hospital and the Aim for the Top University Program of National Taiwan University (grant number 33474, PI: CH Sun). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. There was no additional external funding received for this study.