Cultured human and murine cancer, stem, or immune cell populations
control mechanism
Pooled lentiviral sgRNA transduction at low multiplicity of infection followed by next-generation sequencing (MAGeCK)
pioneer
Feng Zhang lab (GeCKO libraries) and Eric Lander / David Sabatini labs (genome-scale human knockout screens, 2014)
Core Principles & Analysis
Pooled lentiviral libraries such as GeCKO (Genome-Scale CRISPR Knock-Out) deliver 3 to 6 distinct single guide RNAs (sgRNAs) per gene across ~19,000–20,000 protein-coding genes so each transduced cell receives a single gene perturbation.
After applying a selective pressure (such as a targeted anticancer drug, toxin, or viral infection), genomic DNA is amplified by two-step PCR and sequenced by NGS to identify enriched sgRNAs (resistance genes) and depleted sgRNAs (essential or synthetic-lethal genes).
Statistical algorithms such as MAGeCK (Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout, developed in 2014) aggregate fold-changes across multiple sgRNAs targeting the same gene to rank drug targets and synthetic-lethal vulnerabilities.