Analysis of Combinatorial Knockout CRISPR screens with GRAPE: Genetic interaction Regression Analysis of Pairwise Effects

Genetic interactions (GI) reveal functional relationships for understanding gene function and identifying candidate therapeutic vulnerabilities. Combinatorial CRISPR technologies enable genome-scale GI mapping in mammalian cells, but existing analytical methods lack systematic validation against ground truths. We introduce GRAPE (Genetic interaction Regression Analysis of Pairwise Effects), a computational framework that identifies GIs from pooled CRISPR screens by using linear regression to estimate single-gene phenotypes and detecting deviations from expected double-knockout effects. To enable rigorous benchmarking, we developed Synulator, a pipeline that simulates realistic CRISPR screen data with defined synthetic lethal interactions while preserving experimental noise profiles. In simulated screens, GRAPE achieves greater precision and recall compared to existing methods, particularly for interactions with weaker effect sizes. Applying GRAPE to published combinatorial screens across cell lines and CRISPR platforms demonstrates concordance with original findings while identifying additional high-confidence interactions. GRAPE provides a robust, versatile tool for GI mapping, advancing functional genomics and the systematic discovery of synthetic lethal targets in cancer. TeaserA regression-based framework and simulations enable accurate detection of GIs from combinatorial CRISPR screens.