A Novel Screening Approach for the Dissection of Cellular Regulatory Networks of NF-κB Using Arrayed CRISPR gRNA Libraries. (July 2020)

• Record Type: Journal Article
• Authors: O'Shea, Patrick; Wildenhain, Jan; Leveridge, Mathew; Revankar, Chetana; Yang, Jian-Ping; Bradley, Jenna; Firth, Mike; Pilling, James; Piper, David; Chesnut, Jonathan; Isherwood, Beverley
• Is Part Of: SLAS discovery. Volume 25:Number 6(2020)
• Page Start: 618
• Page End: 633
• Publication Date: 2020-07
• Subjects: CRISPR/Cas systems -- functional genomics -- gene library -- cell signaling -- NF-κB; Drugs -- Analysis -- Periodicals; Drugs -- Testing -- Periodicals; Biomolecules -- Analysis -- Periodicals; Biomolecules -- Analysis; Drugs -- Analysis; Drugs -- Testing; Drug Evaluation, Preclinical; Molecular Biology -- methods; Periodicals; Periodicals; 615.1
• DOI: 10.1177/2472555220926160
• Languages: English
• ISSNs: 2472-5552

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.