Abstract

Insulin rapidly represses hepatic glucose production upon feeding. While insulin signals through canonical phosphorylation cascades, the complete set of genes regulating hepatic insulin action is unknown. We developed a genome wide CRISPR screen using fluorescence activated nuclei sorting (FANS) to identify novel regulators of hepatic insulin signalling.

We introduced three genetic modifications in HepG2 cells to facilitate a pooled genome wide CRISPR screen. Firstly, the IGF1 receptor (IGF1R), which is aberrantly expressed in HepG2 cells, was deleted to restore insulin sensitivity. Secondly, CAS9 was stably transduced to allow genome editing. Thirdly, a Forkhead Box Protein O1 (FoxO1)-CLOVER reporter, which is exported from the nucleus upon phosphorylation by Akt1/2, was stably transduced as a readout of insulin signalling. To separate nuclei based on FoxO1-CLOVER content we developed a fluorescence activated nuclei sorting (FANS) protocol.

IGF1R^--FOXO1-CLOVER⁺-Cas9⁺-HepG2 cells were transduced with a genome-wide gRNA library.  Infected cells were treated with insulin and isolated nuclei subjected to FANS based on FoxO1-CLOVER fluorescence. The top (FoxO1-CLOVER^High)and bottom (FoxO1-CLOVER^Low) 10% of the distribution were collected and sequenced for guide representation. As expected, guides targeting the insulin receptor were enriched in the FoxO1-CLOVER^High   population while guides targeting PTEN, a known inhibitor of PI3K-AKT signalling, were overrepresented in the  FoxO1-CLOVER^Low nuclei.

In summary, we have established a novel assay to identify genes regulating insulin-induced FoxO1 nuclear export. This provides a resource for unbiased identification of targets for the treatment of diseases associated with altered insulin signalling, such as type 2 diabetes.