Anvar Nazanin Esmaeili, Lin Chenchu, Ma Xingdi, Wilson Lori L, Steger Ryan, Sangree Annabel K, Colic Medina, Wang Sidney H, Doench John G, Hart Traver
Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
Graduate School of Biomedical Sciences, The University of Texas MD Anderson Cancer Center UTHealth, Houston, TX, USA.
bioRxiv. 2023 Sep 5:2023.01.03.522655. doi: 10.1101/2023.01.03.522655.
Genetic interactions mediate the emergence of phenotype from genotype, but initial technologies for combinatorial genetic perturbation in mammalian cells suffer from inefficiency and are challenging to scale. Recent focus on paralog synthetic lethality in cancer cells offers an opportunity to evaluate different approaches and improve on the state of the art. Here we report a meta-analysis of CRISPR genetic interactions screens, identifying a candidate set of background-independent paralog synthetic lethals, and find that the Cas12a platform provides superior sensitivity and assay replicability. We demonstrate that Cas12a can independently target up to four genes from a single guide array, and we build on this knowledge by constructing a genome-scale library that expresses arrays of four guides per clone, a platform we call 'in4mer'. Our genome-scale human library, with only 49k clones, is substantially smaller than a typical CRISPR/Cas9 monogenic library while also targeting more than four thousand paralog pairs, triples, and quads. Proof of concept screens in four cell lines demonstrate discrimination of core and context-dependent essential genes similar to that of state-of-the-art CRISPR/Cas9 libraries, as well as detection of synthetic lethal and masking/buffering genetic interactions between paralogs of various family sizes, a capability not offered by any extant library. Importantly, the in4mer platform offers a fivefold reduction in the number of clones required to assay genetic interactions, dramatically improving the cost and effort required for these studies.
基因相互作用介导了从基因型到表型的出现，但哺乳动物细胞中组合式基因扰动的初始技术效率低下且难以规模化。最近对癌细胞中旁系同源基因合成致死性的关注提供了一个机会来评估不同方法并改进现有技术水平。在此，我们报告了一项对CRISPR基因相互作用筛选的荟萃分析，确定了一组与背景无关的旁系同源基因合成致死候选基因，并发现Cas12a平台具有更高的灵敏度和检测可重复性。我们证明Cas12a可以从单个向导阵列独立靶向多达四个基因，并且基于这一认识构建了一个基因组规模文库，该文库每个克隆表达四个向导的阵列，我们将这个平台称为“in4mer”。我们的基因组规模人类文库只有49k个克隆，比典型的CRISPR/Cas9单基因文库小得多，同时还能靶向超过四千个旁系同源基因对、三基因组合和四基因组合。在四种细胞系中进行的概念验证筛选表明，它能够区分核心和依赖于背景的必需基因，类似于现有技术水平的CRISPR/Cas9文库，还能检测不同家族规模旁系同源基因之间的合成致死以及掩盖/缓冲基因相互作用，这是任何现有文库都不具备的能力。重要的是，in4mer平台将检测基因相互作用所需的克隆数量减少了五倍，极大地降低了这些研究所需的成本和工作量。
