Broad Institute of MIT and Harvard, 415 Main St., Cambridge, MA 02142, USA; Harvard-MIT Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
Cell. 2022 Nov 23;185(24):4634-4653.e22. doi: 10.1016/j.cell.2022.10.017. Epub 2022 Nov 7.
Understanding the basis for cellular growth, proliferation, and function requires determining the roles of essential genes in diverse cellular processes, including visualizing their contributions to cellular organization and morphology. Here, we combined pooled CRISPR-Cas9-based functional screening of 5,072 fitness-conferring genes in human HeLa cells with microscopy-based imaging of DNA, the DNA damage response, actin, and microtubules. Analysis of >31 million individual cells identified measurable phenotypes for >90% of gene knockouts, implicating gene targets in specific cellular processes. Clustering of phenotypic similarities based on hundreds of quantitative parameters further revealed co-functional genes across diverse cellular activities, providing predictions for gene functions and associations. By conducting pooled live-cell screening of ∼450,000 cell division events for 239 genes, we additionally identified diverse genes with functional contributions to chromosome segregation. Our work establishes a resource detailing the consequences of disrupting core cellular processes that represents the functional landscape of essential human genes.
要了解细胞生长、增殖和功能的基础，需要确定在各种细胞过程中基本基因的作用，包括观察它们对细胞组织和形态的贡献。在这里，我们将 5072 个人类 HeLa 细胞中与功能相关的 CRISPR-Cas9 基因进行了基因敲除，同时结合显微镜下观察 DNA、DNA 损伤反应、肌动蛋白和微管，对超过 3100 万个细胞进行了分析。对 >90%的基因敲除细胞的表型进行了可测量的分析，这些表型涉及特定的细胞过程，表明基因靶标与特定的细胞过程相关。基于数百个定量参数的表型相似性聚类，进一步揭示了不同细胞活动中的共功能基因，为基因功能和关联提供了预测。通过对 239 个基因的约 450000 个细胞分裂事件进行基因敲除的细胞活筛，我们还鉴定出了在染色体分离过程中有功能贡献的不同基因。我们的工作建立了一个详细描述破坏核心细胞过程的后果的资源，代表了人类必需基因的功能图谱。
