Combinatorial genetic screening using CRISPR-Cas9 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 used machine learning to establish S. aureus Cas9 sgRNA design rules and paired S. aureus Cas9 with S. pyogenes 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. The "Big Papi" (paired aureus and pyogenes for interactions) approach described here will be widely applicable for the study of combinatorial phenotypes.

中文翻译：

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用于组合遗传筛选的直系同源 CRISPR-Cas9 酶。


使用 CRISPR-Cas9 的组合遗传筛选是发现冗余基因和探索复杂基因网络的有用方法。然而，目前的方法受到单引导 RNA (sgRNA) 之间的干扰以及有限的基因靶向活性的影响。为了提高组合筛选的效率，我们采用来自金黄色葡萄球菌和化脓性链球菌的正交 Cas9 酶。我们利用机器学习建立了金黄色葡萄球菌 Cas9 sgRNA 设计规则，并将金黄色葡萄球菌 Cas9 与化脓性链球菌 Cas9 配对，以在大部分细胞中实现双重靶向。我们还开发了慢病毒载体和克隆策略，以生成高复杂性的混合双敲除文库，以识别多种细胞类型中的合成致死和缓冲基因对，包括 MAPK 途径基因和凋亡基因。我们的直系同源方法还实现了将基因敲除与转录激活相结合的筛选，揭示了与 TP53 的遗传相互作用。这里描述的“Big Papi”（配对金黄色葡萄球菌和化脓菌相互作用）方法将广泛适用于组合表型的研究。