Integrated arrayed CRISPR pipeline to mechanistically annotate sensitisation hits to PARP inhibitors

Integrated pooled and arrayed CRISPR screening can be used to identify and mechanistically characterise genes that confer drug resistance and sensitivity. PARP inhibitors have demonstrated clinical efficacy in cancers with defects in the DNA homologous recombination repair pathway, such as BRCAm cancers. Efforts are continuing to identify further patient populations that may receive therapeutic benefit from PARP inhibition. Here we describe the development of a high content imaging assay used to screen 100’s gene knockouts (KOs) identified in a pooled CRISPR screen as sensitisers to PARP inhibition, to provide deeper mechanistic insights into the cellular response to drug sensitisation.
Using the colorectal cancer cell line DLD1 stably expressing Cas9 and a lipid-based transfection approach, we knocked out ~200 sensitiser genes identified in genome wide screens . To assess potential mechanisms underlying this response, we developed an immunofluorescence assay measuring multiple DNA damage and cell cycle markers (γH2AX, 53BP1 foci, micronuclei, Hoescht intensity). Using this approach and a statistical workflow, we developed a robust and reliable HTS compatible assay that enabled hit selection based on the effects of individual gene KO +/- PARP inhibition. Based on this we clustered gene KOs based on their ‘molecular phenotype’, helping us to prioritise those hits to move forward to a high throughput proliferation assay for further confirmation of the sensitisation effect.
In summary, our integrated CRISPR pipeline has enabled to identification and prioritisation of PARPi sensitisers, helping us work towards identification of novel patient populations that may benefit from this therapeutic approach.