Abstract

At the Medicines Discovery Catapult (MDC), we work in partnership with UK businesses and academics to progress their projects towards clinical application. Our science strategy is focused on complex cell models, target engagement and validation, pre-clinical imaging and biomarkers. The ability to manipulate target proteins and measure the biological consequences of such a change is an important objective. The application of CRISPR/Cas genome editing to knock-out endogenous cellular proteins is established as a key methodology for in vitro disease modelling and target validation, therefore aligning closely to the MDC science strategy. We have established workflows to generate and validate CRISPR/Cas knock-out cell lines and subsequently to characterize these cellular models using a range of molecular and cell biology techniques, including microscopy and single-cell transcriptomics. In these studies, we have used as a model system a commercial A549 lung adenocarcinoma cell line, modified to encode GFP at the 3’ end of the epidermal growth factor receptor (EGFR) gene. To achieve knock-out, a modified single guide sgRNA was designed to target the 2^nd exon of EGFR and, following transfection and selection, digital droplet PCR (ddPCR) was applied to determine the efficiency of editing and to identify homozygous knockout clones. These clones were subsequently characterized by Western blot and advanced microscopy. To measure the downstream effects of EGFR knock-out, single-cell RNAseq was applied using the 10x genomics platform in conjunction with next-generation sequencing. In the future, we plan to apply the workflows described above facilitate the development of in vitro cellular models of disease and ultimately to support the identification and progression of new drug targets.