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Updated: May 27, 2025
Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells
Published on: June 16, 2017
A genome-wide CRISPR-Cas9 knockout screen using dynamin knockout cells identifies Nf2 and Traf3 as genes involved in
Sho Takahashi1, Mizuho Maehara1, Chihiro Nishihara1
1Laboratory of Veterinary Hygiene, Joint Faculty of Veterinary Medicine, Yamaguchi University, 1677-1, Yoshida, Yamaguchi, 753-8515, Japan.
Abstract:
Endocytosis is a fundamental process by which cells take up extracellular materials, including nutrients, growth factors, and pathogens. Although several endocytic pathways, such as clathrin-mediated and caveolin-mediated endocytosis, are well-characterized, other endocytic pathways remain poorly understood. Therefore, in this study, we performed a genome-wide CRISPR-Cas9 screen to elucidate new endocytic pathways using dynamin conditional knockout cells. We identified genes that significantly reduced the cell numbers when knocked out simultaneously with dynamin. Among these, neurofibromin 2 (Nf2) and tumor necrosis factor receptor-associated factor 3 (Traf3), whose relationship with endocytosis was not well understood, were investigated for their roles in endocytosis activity. Nf2 and Traf3 knockout cells exhibited reduced non-specific fluid endocytosis in a dynamin-independent manner. However, Nf2 or Traf3 knockout did not affect the transferrin receptor-mediated endocytosis that depends on clathrin and dynamin. Moreover, Nf2 knockout cells showed reduced cholera toxin uptake in a dynamin-independent manner. Overall, this study highlights the roles of Nf2 and Traf3 in endocytosis.
Insights
This study reveals new roles for neurofibromin 2 (Nf2) and tumor necrosis factor receptor-associated factor 3 (Traf3) in cellular uptake processes. Knocking out these genes impairs non-specific endocytosis independently of dynamin.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- Endocytosis is crucial for cellular uptake of various materials.
- While clathrin-mediated and caveolin-mediated endocytosis are known, other pathways remain unclear.
- Dynamin is a key protein involved in multiple endocytic pathways.
Purpose of the Study:
- To identify novel genes involved in endocytosis using a genome-wide CRISPR-Cas9 screen.
- To investigate the roles of neurofibromin 2 (Nf2) and tumor necrosis factor receptor-associated factor 3 (Traf3) in endocytic mechanisms.
- To elucidate the relationship between Nf2, Traf3, and dynamin-dependent/independent endocytosis.
Main Methods:
- Genome-wide CRISPR-Cas9 screening in dynamin conditional knockout cells.
- Analysis of gene knockouts affecting cell viability.
- Assays for non-specific fluid endocytosis and transferrin receptor-mediated endocytosis.
- Cholera toxin uptake experiments in Nf2 knockout cells.
Main Results:
- Identified Nf2 and Traf3 as genes crucial for cell survival when dynamin is absent.
- Nf2 and Traf3 knockout cells showed reduced non-specific fluid endocytosis, independent of dynamin.
- Transferrin receptor-mediated endocytosis, dependent on clathrin and dynamin, was unaffected by Nf2 or Traf3 knockout.
- Nf2 knockout cells exhibited decreased cholera toxin uptake in a dynamin-independent manner.
Conclusions:
- Nf2 and Traf3 play significant roles in specific endocytic pathways.
- These proteins are involved in dynamin-independent endocytosis.
- The findings expand our understanding of the complex mechanisms governing cellular uptake.
