A Novel Screening Approach for the Dissection of Cellular Regulatory Networks of NF-κB Using Arrayed CRISPR gRNA Libraries

SLAS Discov. 2020 Jul;25(6):618-633. doi: 10.1177/2472555220926160. Epub 2020 Jun 1.

Abstract

CRISPR/Cas9 is increasingly being used as a tool to prosecute functional genomic screens. However, it is not yet possible to apply the approach at scale across a full breadth of cell types and endpoints. In order to address this, we developed a novel and robust workflow for array-based lentiviral CRISPR/Cas9 screening. We utilized a β-lactamase reporter gene assay to investigate mediators of TNF-α-mediated NF-κB signaling. The system was adapted for CRISPR/Cas9 through the development of a cell line stably expressing Cas9 and application of a lentiviral gRNA library comprising mixtures of four gRNAs per gene. We screened a 743-gene kinome library whereupon hits were independently ranked by percent inhibition, Z' score, strictly standardized mean difference, and T statistic. A consolidated and optimized ranking was generated using Borda-based methods. Screening data quality was above acceptable limits (Z' ≥ 0.5). In order to determine the contribution of individual gRNAs and to better understand false positives and negatives, a subset of gRNAs, against 152 genes, were profiled in singlicate format. We highlight the use of known reference genes and high-throughput, next-generation amplicon and RNA sequencing to assess screen data quality. Screening with singlicate gRNAs was more successful than screening with mixtures at identifying genes with known regulatory roles in TNF-α-mediated NF-κB signaling and was found to be superior to previous RNAi-based methods. These results add to the available data on TNF-α-mediated NF-κB signaling and establish a high-throughput functional genomic screening approach, utilizing a vector-based arrayed gRNA library, applicable across a wide variety of endpoints and cell types at a genome-wide scale.

Keywords: CRISPR/Cas systems; NF-κB; cell signaling; functional genomics; gene library.

MeSH terms

  • CRISPR-Cas Systems / genetics*
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics*
  • Gene Library
  • Genes, Reporter / genetics
  • Genome, Human / genetics
  • High-Throughput Screening Assays / methods
  • Humans
  • NF-kappa B / genetics*
  • Phosphotransferases / classification
  • Phosphotransferases / genetics
  • RNA, Guide, CRISPR-Cas Systems / genetics
  • Signal Transduction / genetics
  • Tumor Necrosis Factor-alpha / genetics*
  • beta-Lactamases / genetics

Substances

  • NF-kappa B
  • RNA, Guide, CRISPR-Cas Systems
  • Tumor Necrosis Factor-alpha
  • Phosphotransferases
  • beta-Lactamases