A high-throughput arrayed CRISPR/Cas9 functional genomics approach to study NF-kB signaling
Recorded On: 02/06/2018
CRISPR/Cas9 is increasingly being used as a tool to prosecute functional genomic screens. Array-based CRISPR/Cas9 screens offer the ability to interrogate more diverse phenotypes than pool-based screens but their execution at scale in difficult to transfect cells brings challenges. We undertook an array-based CRISPR/Cas9 screen at 800 kinome-scale to investigate mediators of TNFα-mediated NF-κB activation. We used an ME180 cell line stably expressing Cas9 and a beta-lactamase reporter of Nf-κB activation alongside a developed lentiviral sgRNA library. Hits were validated by confirmation of DNA insertion/deletion and screening orthogonal reagents. Screening data quality was within acceptable limits (Z’>0.6) and genes associated with canonical NF-κB signalling were identified. Our data provide unique insights into approaches and tools to explore novel biology with array-based gene editing in cellular assays. Our results demonstrate the potential for genome-scale screens at high specificity using CRISPR/Cas9 and in a wide variety of cell backgrounds and phenotypes.
Patrick O'Shea is a Senior Scientist in the High Content Biology group within Discovery Sciences at AstraZeneca in Cambridge UK; a multidisciplinary R&D unit with a global remit. The team develops novel phenotypic assays and uses high-content and functional genomic approaches to understand novel biology, provide an early insight into compound toxicity, and to characterize compound mechanism of action in disease relevant models. Prior to joining AstraZeneca, Patrick studied Pharmacology at the University of Leeds. He completed a PhD at Imperial College London, studying the developmental effects of thyroid hormones in bone and cartilage, and was a postdoctoral fellow at Imperial College London and at the National Cancer Institute, NIH.