A time-resolved imaging-based CRISPRi screening method

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. Specifically, this has restricted studies of intracellular dynamics to one strain at a time and thus, generally, to the impact of genes with known function. Here we show how in situ genotyping of a library of E. coli strains after time-lapse imaging in a microfluidic device overcomes this problem. We determine how 235 different CRISPR interference (CRISPRi) knockdowns impact the coordination of the replication and division cycles of E. coli by monitoring the location of replication forks throughout on average >500 cell cycles per knockdown. The single-cell time-resolved assay allows us to determine the distribution of single-cell growth rates, cell division sizes, and replication initiation volumes. Subsequent in situ genotyping allows us to map each phenotype distribution to a specific genetic perturbation in order to determine which genes are important for cell cycle control. The technology presented in this study enables genome-scale screens of virtually all live-cell microscopy assays and, therefore, constitutes a qualitatively new approach to cellular biophysics.