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

Area of Science:

• Genomics
• Molecular Biology
• Cell Biology

Background:

• CRISPR/Cas9 is a powerful tool for functional genomic screens.
• Current limitations hinder its large-scale application across diverse cell types and endpoints.
• Investigating TNF-α-mediated NF-κB signaling requires robust screening methods.

Purpose of the Study:

• To develop a novel, robust, and scalable workflow for array-based lentiviral CRISPR/Cas9 screening.
• To identify mediators of TNF-α-mediated NF-κB signaling using this new screening approach.
• To establish a high-throughput functional genomic screening platform applicable across various cell types and endpoints.

Main Methods:

• Developed a lentiviral CRISPR/Cas9 screening workflow using a β-lactamase reporter gene assay.
• Created a cell line stably expressing Cas9 and utilized a lentiviral gRNA library.
• Screened a 743-gene kinome library, employing Borda-based methods for consolidated hit ranking.
• Assessed screen quality using reference genes and next-generation sequencing.

Main Results:

• The developed CRISPR/Cas9 screening system demonstrated high data quality (Z' ≥ 0.5).
• Screening with singlicate guide RNAs (gRNAs) was more effective than using gRNA mixtures.
• Identified known regulatory genes in TNF-α-mediated NF-κB signaling with superior performance compared to RNAi methods.

Conclusions:

• Established a high-throughput, array-based CRISPR/Cas9 screening approach for functional genomics.
• The workflow is applicable to a wide range of cellular endpoints and cell types at a genome-wide scale.
• This method provides a significant advancement for large-scale functional genomic studies.