CRISPR Screening: Molecular Tools for Studying Virus–Host Interactions

Figures

• 

  Figure 1. General workflow of CRISPR screens. CRISPR screening protocols include 6 main steps: (1) in silico design of gRNA libraries; (2) cloning and validation of gRNA library; (3) packaging gRNAs into lentiviruses and transduction of lentiviruses into Cas-expressing cells; (4) alteration of genes in experimental conditions; (5) isolation of nucleic acids; and (6) deep sequencing of barcoded gRNAs and data analysis. Note: if using pre-made gRNA libraries, the first two steps of the protocol are omitted.

• 

  Figure 2. Positive and negative selection of cells for CRISPR screening. Cas-producing cells are transduced with the lentiviral gRNA library. Next, cells are infected with a cytolytic virus and then undergo positive selection (resistant cells are enriched while susceptible cells die) or negative selection (cells with altered antiviral genes die).