Combinatorial genetic screening using CRISPR\u2013Cas9 is a useful approach to uncover redundant genes and to explore complex gene networks. However, current methods suffer from interference between the single-guide RNAs (sgRNAs) and from limited gene targeting activity. To increase the efficiency of combinatorial screening, we employ orthogonal Cas9 enzymes from Staphylococcus aureus<\/i> and Streptococcus pyogenes<\/i>. We used machine learning to establish S. aureus<\/i> Cas9 sgRNA design rules and paired S. aureus<\/i> Cas9 with S. pyogenes<\/i> Cas9 to achieve dual targeting in a high fraction of cells. We also developed a lentiviral vector and cloning strategy to generate high-complexity pooled dual-knockout libraries to identify synthetic lethal and buffering gene pairs across multiple cell types, including MAPK pathway genes and apoptotic genes. Our orthologous approach also enabled a screen combining gene knockouts with transcriptional activation, which revealed genetic interactions with TP53<\/i>. The \u201cBig Papi\u201d (paired aureus<\/i> and pyogenes<\/i> for interactions) approach described here will be widely applicable for the study of combinatorial phenotypes.<\/p>\n","protected":false},"excerpt":{"rendered":"
Combinatorial genetic screening using CRISPR\u2013Cas9 is a useful approach to uncover redundant genes and to explore complex gene networks. However, current methods suffer from interference between the single-guide RNAs (sgRNAs) and from limited gene targeting activity. To increase the efficiency of combinatorial screening, we employ orthogonal Cas9 enzymes from Staphylococcus aureus and Streptococcus pyogenes. We 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