Orthologous CRISPR-Cas9 for Combinatorial Genetic Screens

CRISPR-Cas9 has enabled a new generation of screening strategies to interrogate gene function. Single gene knockout approaches, however, encounter limitations in exploring redundant genes and complex gene networks. Here we enable efficient combinatorial screens by combining orthogonal CRISPR-derived components to avoid interference and maximize gene targeting activity. We used machine learning to establish efficient S. aureus Cas9 sgRNA design rules, which we paired with S. pyogenes Cas9 to achieve dual target gene inactivation in a high fraction of cells. We developed a lentiviral vector and cloning strategy to generate high complexity pooled dual-knockout libraries and identify synthetic lethal gene pairs across multiple cell types, including MAPK pathway genes and anti-apoptotic genes. Notably, our orthologous approach permits more flexible manipulations using distinct Cas9 modalities for each gene, such as knockout plus transcriptional activation. The “Big Papi” approach described here will be widely applicable for the study of combinatorial phenotypes.