A genome-wide CRISPR-Cas9 knockout screen using dynamin knockout cells identifies Nf2 and Traf3 as genes involved in dynamin-independent endocytosis.

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.