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MBE CRISPR screen disease gene factor

1. Experimental Purpose

  • Scientific Question: Which human genes are essential for virus X infection and replication?
  • Design Rationale: Genome-wide CRISPR screen enables identification of host dependency factors required for viral entry, replication, assembly, or release
  • Follow-up Studies: Investigate mechanism of top hits, develop small molecule inhibitors targeting key host factors, test for broad-spectrum activity against related viruses

2. Model System

  • Primary System: Human cell line susceptible to virus X infection (e.g., Huh7 cells for hepatitis C virus)
  • Rationale: Permissive cell lines that support complete viral lifecycle while maintaining high Cas9 editing efficiency provide ideal platform for identifying host factors
  • Alternatives:
  • Primary human cells (pros: physiological relevance; cons: difficult to scale, variable editing)
  • Immortalized cells from target organs (pros: tissue-specific factors; cons: may have altered cellular pathways)
  • Reporter virus systems (pros: biosafety, high-throughput; cons: may miss late-stage factors)
  • Ethical Considerations: Biosafety protocols for viral work, cell line authentication, appropriate containment levels

3. Measurement Approach

  • Techniques:
  • Lentiviral delivery of genome-wide sgRNA library to Cas9-expressing cells
  • Viral challenge with optimized MOI
  • Cell survival/death assay or FACS-based sorting of infected vs. uninfected cells
  • Next-generation sequencing of sgRNA abundance pre/post-selection
  • Technical Replicates: Duplicate NGS library preparations, multiple infection timepoints
  • Potential Biases:
  • Viral stock variations (standardize preparation, titration)
  • Infection efficiency differences (optimize viral MOI)
  • Cell death from Cas9 toxicity (include essential gene controls)
  • Off-target effects (use libraries with minimal predicted off-targets)

4. Group Setting

  • Experimental Groups:
  • Test: sgRNA library-transduced cells infected with virus X
  • Control 1: Uninfected sgRNA library cells (baseline sgRNA distribution)
  • Control 2: Non-targeting sgRNA library with viral infection
  • Control 3: Time-matched uninfected cells for growth-related effects
  • Controlled Variables: Cell density, viral dose, infection duration, sgRNA library coverage
  • Biological Replicates: 3-4 independent library transductions and viral challenges
  • Modified Design: Include CRISPR screens with multiple viral strains to identify strain-specific vs. conserved host factors

5. Data Analysis & Presentation

  • Data Processing:
  • sgRNA read counting from sequencing data
  • Normalization to library depth
  • Enrichment/depletion calculation relative to uninfected controls
  • Gene-level significance using MAGeCK or similar algorithms
  • Statistical Analysis:
  • False discovery rate control for multiple testing
  • Comparison with published essential gene datasets
  • Pathway and network analysis of hits
  • Comparison with known viral interactors from proteomics data
  • Data Presentation:
  • Manhattan plots of gene significance across the genome
  • Cellular pathway maps highlighting hit clusters
  • Validation data for top hits showing infection rates with individual knockouts
  • Comparison with previous genetic screens or interactome studies
  • Validation Methods:
  • Individual gene knockout confirmation
  • Viral replication assays with knockout cell lines
  • Rescue experiments with cDNA expression
  • Protein-protein interaction studies with viral components
  • Small molecule inhibition of identified pathways