Loss-of-function and gain-of-function genetic perturbations provide valuable insights into gene function. In 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 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 (), a member of the lipocalin protein family (), and a zinc finger C2H2 transcription factor (). Mechanistically, we demonstrate that overexpression activates the RTK-Akt-mTOR signaling pathway and that activation of insulin receptor (InR) by requires cholesterol and clathrin-coated pits at the cell membrane. This study establishes a novel platform for functional genetic studies in cells.
Genome-wide screen
2023
Feb
24
2023
Feb
17
A genome-wide CRISPR screen identifies DPM1 as a modifier of DPAGT1 deficiency and ER stress.” PLoS Genet, 18, 9, Pp. e1010430.Abstract
. 2022. “
CRISPR screens in Drosophila cells identify Vsg as a Tc toxin receptor.” Nature, 610, 7931, Pp. 349-355.Abstract
. 2022. “
State-of-the-art CRISPR for in vivo and cell-based studies in Drosophila.” Trends Genet, 38, 5, Pp. 437-453.Abstract
. 2022. “So you want to do a CRISPR pooled screen in insect cells? You can! Here's how
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Pooled CRISPR Screens in Drosophila Cells.” Curr Protoc Mol Biol, 129, 1, Pp. e111.Abstract
. 2019. “DRSC-Biomedical Technology Research Resource
A Membrane Transporter Is Required for Steroid Hormone Uptake in Drosophila.” Dev Cell, 47, 3, Pp. 294-305.e7.Abstract
. 2018. “