Institute of Biochemistry II, Faculty of Medicine, Goethe University Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany.
Department of Medicine, Hematology/Oncology, University Hospital, Goethe University, 60590 Frankfurt am Main, Germany.
Nucleic Acids Res. 2021 Jun 4;49(10):5684-5704. doi: 10.1093/nar/gkab309.
Combinatorial CRISPR-Cas screens have advanced the mapping of genetic interactions, but their experimental scale limits the number of targetable gene combinations. Here, we describe 3Cs multiplexing, a rapid and scalable method to generate highly diverse and uniformly distributed combinatorial CRISPR libraries. We demonstrate that the library distribution skew is the critical determinant of its required screening coverage. By circumventing iterative cloning of PCR-amplified oligonucleotides, 3Cs multiplexing facilitates the generation of combinatorial CRISPR libraries with low distribution skews. We show that combinatorial 3Cs libraries can be screened with minimal coverages, reducing associated efforts and costs at least 10-fold. We apply a 3Cs multiplexing library targeting 12,736 autophagy gene combinations with 247,032 paired gRNAs in viability and reporter-based enrichment screens. In the viability screen, we identify, among others, the synthetic lethal WDR45B-PIK3R4 and the proliferation-enhancing ATG7-KEAP1 genetic interactions. In the reporter-based screen, we identify over 1,570 essential genetic interactions for autophagy flux, including interactions among paralogous genes, namely ATG2A-ATG2B, GABARAP-MAP1LC3B and GABARAP-GABARAPL2. However, we only observe few genetic interactions within paralogous gene families of more than two members, indicating functional compensation between them. This work establishes 3Cs multiplexing as a platform for genetic interaction screens at scale.
组合型 CRISPR-Cas 筛选技术已经推进了遗传相互作用的研究，但由于其实验规模限制了靶向基因组合的数量。在这里，我们描述了 3Cs 多重化技术，这是一种快速且可扩展的方法，可以生成高度多样化且均匀分布的组合型 CRISPR 文库。我们证明了文库分布偏度是决定其所需筛选覆盖范围的关键决定因素。通过避免 PCR 扩增寡核苷酸的迭代克隆，3Cs 多重化技术有助于生成具有低分布偏度的组合型 CRISPR 文库。我们表明，组合型 3Cs 文库可以用最小的覆盖范围进行筛选，至少将相关工作和成本降低 10 倍。我们应用了一个靶向 12736 个自噬基因组合、包含 247032 对靶向 gRNA 的 3Cs 多重化文库，在基于活力和报告基因的富集筛选中进行了实验。在活力筛选中，我们除其他外，鉴定了合成致死性 WDR45B-PIK3R4 和增殖增强性 ATG7-KEAP1 遗传相互作用。在基于报告基因的筛选中，我们鉴定了超过 1570 个自噬通量的必需遗传相互作用，包括同源基因之间的相互作用，例如 ATG2A-ATG2B、GABARAP-MAP1LC3B 和 GABARAP-GABARAPL2。然而，我们仅在具有两个以上成员的同源基因家族内观察到少数遗传相互作用，这表明它们之间存在功能补偿。这项工作确立了 3Cs 多重化技术作为大规模遗传相互作用筛选的平台。
