Assaying for RNA abundance and protein phosphorylation in pooled CRISPR screens

Pooled CRISPR screens are a powerful tool to identify regulators of a biological process, but have been limited to phenotypes that affect viability or can be monitored with fluorescent protein reporters. We evaluate two additional strategies for phenotypic enrichment based on quantification of RNA using a Flow-FISH assay or protein phosphorylation through intracellular phospho-staining. These tools will greatly expand the applicability of CRISPR screens since they increase the number of possible molecular phenotypes and assay designs. Overall design: Pooled Knockout CRISPR screen comparing Nanog Flow-FISH vs a C-terminally tagged mCherry Nanog reporter and an intracellular pMek-staining vs a live MAPK GFP reporter as readout systems. An sgRNA library targeting annotated phosphatases is implemented.