Abstract

Functional genomics approaches such as pooled and arrayed CRISPR screening have revolutionised drug and target discovery. Pooled CRISPR screens coupled with gRNA sequencing readouts are widely adopted for genome wide assessment of genes and pathways involved in drug sensitisation and resistance. However, in order to identify the most promising hits for follow up study, an orthogonal growth assay is required to validate primary screening results. Here we describe the work we have done to build a high-throughput long-term proliferation assay coupled with an imaging readout to follow up targets identified by genome wide pooled CRISPR screening as sensitisers to small molecule inhibitors. Specifically, we created an automated workflow allowing counting and seeding of two cell backgrounds in parallel in 96-well plates, after infection with lentiviruses expressing gRNAs targeting selected genes of interest. Infected cells were then treated with the drug for 14 days and their proliferation was monitored using an Incucyte. This work has the potential to greatly increase the capacity for running long-term proliferation assays, enabling the validation at scale of oncology targets identified by pooled CRISPR screening. Most importantly, our proof of concept validation screen facilitated the prioritisation of selected novel sensitivity targets, based on the strength and reproducibility of the observed proliferation phenotype, ultimately informing patient-selection strategies.