Cellular Technologies

A high-throughput arrayed CRISPR/Cas9 functional genomics approach to study NF-kB signaling

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  Patrick O'Shea, PhD

  Senior Research Scientist
  Astrazeneca

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.