Pooled genome-wide CRISPR activation screening for rapamycin resistance genes in <i>Drosophila</i> cells.

Abstract

Loss-of-function and gain-of-function genetic perturbations provide valuable insights into gene function. In <i>Drosophila</i> cells, while genome-wide loss-of-function screens have been extensively used to reveal mechanisms of a variety of biological processes, approaches for performing genome-wide gain-of-function screens are still lacking. Here, we describe a pooled CRISPR activation (CRISPRa) screening platform in <i>Drosophila</i> cells and apply this method to both focused and genome-wide screens to identify rapamycin resistance genes. The screens identified three genes as novel rapamycin resistance genes: a member of the SLC16 family of monocarboxylate transporters (<i>CG8468</i>), a member of the lipocalin protein family (<i>CG5399</i>), and a zinc finger C2H2 transcription factor (<i>CG9932</i>). Mechanistically, we demonstrate that <i>CG5399</i> overexpression activates the RTK-Akt-mTOR signaling pathway and that activation of insulin receptor (InR) by <i>CG5399</i> requires cholesterol and clathrin-coated pits at the cell membrane. This study establishes a novel platform for functional genetic studies in <i>Drosophila</i> cells.