A Three-Way Combinatorial CRISPR Screen for Analyzing Interactions among Druggable Targets

We present a CRISPR-based multi-gene knockout screening system and toolkits for extensible assembly of barcoded high-order combinatorial guide RNA libraries en masse. We apply this system for systematically identifying not only pairwise but also three-way synergistic therapeutic target combinations and successfully validate double- and triple-combination regimens for suppression of cancer cell growth and protection against Parkinson’s disease-associated toxicity. This system overcomes the practical challenges of experimenting on a large number of high-order genetic and drug combinations and can be applied to uncover the rare synergistic interactions between druggable targets. [Display omitted] •Toolkits are engineered for extensible assembly of barcoded high-order gRNA arrays•Simultaneous knockout of up to three genes for combinatorial CRISPR screening•CRISPR screens identify infrequent synergistic interactions among druggable targets•Gene interactions are translated to matching drugs for disease phenotype modulation Studying high-order druggable gene interactions that mimic the actions of targeted drug combinations is practically challenging. Zhou et al. describe an extensible CombiGEM-CRISPR system for screening high-order combinations, leading to identification of double- and triple-combination regimens that suppress ovarian cancer growth and Parkinson’s disease-associated toxicity.