In this work, we present a proof-of-principle experiment that extends advanced live cell microscopy to the scale of pool-generated strain libraries. We achieve this by identifying the genotypes for individual cells in situ after a detailed characterization of the phenotype. The principle is demonstrated by single-molecule fluorescence time-lapse imaging of Escherichia coli strains harboring barcoded plasmids that express a sgRNA which suppresses different genes in the E. coli genome through dCas9 interference. In general, the method solves the problem of characterizing complex dynamic phenotypes for diverse genetic libraries of cell strains. For example, it allows screens of how changes in regulatory or coding sequences impact the temporal expression, location, or function of a gene product, or how the altered expression of a set of genes impacts the intracellular dynamics of a labeled reporter.

中文翻译：

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在表征复杂表型后对混合菌株库进行原位基因分型。


在这项工作中，我们提出了一个原理验证实验，将先进的活细胞显微镜扩展到池生成的菌株库的规模。我们通过在详细表征表型后原位鉴定单个细胞的基因型来实现这一目标。该原理通过对含有条形码质粒的大肠杆菌菌株进行单分子荧光延时成像得到证实，这些质粒表达 sgRNA，该 sgRNA 通过 dCas9 干扰抑制大肠杆菌基因组中的不同基因。一般来说，该方法解决了表征不同细胞株遗传文库的复杂动态表型的问题。例如，它可以筛选调控序列或编码序列的变化如何影响基因产物的时间表达、位置或功能，或者一组基因的表达改变如何影响标记报告基因的细胞内动态。