Time-resolved imaging-based CRISPRi screening
Time-resolved imaging-based CRISPRi screening
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DOI：
10.1038/s41592-019-0629-y
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发表时间：
2020-01-01
期刊：
影响因子：
48
通讯作者：
Elf, Johan
中科院分区：
文献类型：
--
作者：
Camsund, Daniel;Lawson, Michael J.;Elf, Johan
DuMPLING（在原位基因分型之前对库进行动态多流控显微镜表型分析）能够筛选菌株库中的动态表型，并在这里用于研究协调大肠杆菌复制和细胞分裂的基因。我们将基因型变异与生物学重要表型联系起来的能力受到活细胞显微镜和库规模基因组工程之间的差距的严重限制。在这里，我们展示了如何在微流控装置中延时成像后对菌株库进行原位基因分型，从而克服了这个问题。我们确定了235种不同的CRISPR干扰敲除如何影响大肠杆菌复制和分裂周期的协调，通过监测复制叉的位置，平均每个敲除>500个细胞周期。随后的原位基因分型使我们能够将每个表型分布映射到特定的遗传扰动，以确定哪些基因对细胞周期控制很重要。单细胞时间分辨测定允许我们确定单细胞生长速率、细胞分裂大小和复制起始体积的分布。这项研究中提出的技术使大多数活细胞显微镜检测的基因组规模的屏幕。
DuMPLING (dynamic mu-fluidic microscopy phenotyping of a library before in situ genotyping) enables screening of dynamic phenotypes in strain libraries and was used here to study genes that coordinate replication and cell division in Escherichia coli.Our ability to connect genotypic variation to biologically important phenotypes has been seriously limited by the gap between live-cell microscopy and library-scale genomic engineering. Here, we show how in situ genotyping of a library of strains after time-lapse imaging in a microfluidic device overcomes this problem. We determine how 235 different CRISPR interference knockdowns impact the coordination of the replication and division cycles of Escherichia coli by monitoring the location of replication forks throughout on average >500 cell cycles per knockdown. Subsequent in situ genotyping allows us to map each phenotype distribution to a specific genetic perturbation to determine which genes are important for cell cycle control. The single-cell time-resolved assay allows us to determine the distribution of single-cell growth rates, cell division sizes and replication initiation volumes. The technology presented in this study enables genome-scale screens of most live-cell microscopy assays.