Pooled genome-wide CRISPR activation screening for rapamycin resistance genes inDrosophilacells

Abstract

Loss-of-function and gain-of-function genetic perturbations provide valuable insights into gene function. In Drosophila 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 Drosophila 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 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 that CG5399 overexpression activates the RTK-Akt-mTOR signaling pathway and that activation of InR by CG5399 requires cholesterol and clathrin-coated pits at the cell membrane. This study establishes a novel platform for functional genetic studies in Drosophila cells.

Genetic manipulation of anIxodes scapulariscell line

“…Specifically, CRISPRa might be considered a valuable tool for orthogonally validating immunity and fitness studies in I. scapularis . By over-expressing genes of interest in I. scapularis cells, one may complement RNAi-based technologies and develop small- or large-scale studies, such as commonly reported genome- wide pooled CRISPRa screens in mammals and Drosophila (28, 30).…”