Loss-of-function and gain-of-function genetic perturbations provide valuable insights into gene function. InDrosophilacells, 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 inDrosophilacells 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 SLC16 family of monocarboxylate transporters (CG8468), a member of the lipocalin protein family (CG5399), and a zinc finger C2H2 transcription factor (CG9932). Mechanistically, we demonstrate thatCG5399overexpression activates the RTK-Akt-mTOR signaling pathway and that activation of InR byCG5399requires cholesterol and clathrin-coated pits at the cell membrane. This study establishes a novel platform for functional genetic studies inDrosophilacells.
