Abstract

Integrated pooled and arrayed CRISPR screening can be used to identify and mechanistically characterise genes that confer drug resistance and sensitivity. Here we describe coupling cell cycle analysis using Hoechst staining into a high content imaging assay used to screen 100’s gene KOs identified in genome pooled screens as sensitisers to PARP inhibition. Using the colorectal cancer cell line DLD1 stably expressing Cas9 and a lipid-based transfection approach we knocked-out ~200 genes selected from whole genome CRISPR screens and treated with PARP inhibitors. Applying Hoechst staining, we measured the amount of DNA in the cell nuclei using high content imaging, allowing us to determine the fraction of cells in each of the cell cycle phases. Statistical comparison of gene KO wells compared with the neutral controls determined significant changes in the cell cycle phase distribution, in the presence and absence of PARP inhibitors, identified gene KOs that result in cell cycle modulation as a result of DNA damage accumulation. Characterisation of the cell cycle arrest information has helped further annotate hits and improve our understanding of DNA damage accumulation mechanisms upon PARP inhibition and will be integrated into additional endpoints determined by arrayed CRISPR screening.