Figure 1.

Summary of experimental workflow. Multiple inserts including optimised mEos variants were cloned into identical donor plasmids to assess the effects of fluorophore properties on endogenous expression after CRISPR knock-in. Cells were identically transfected with specific guides, and after the clonal isolation of the brightest (and hence best expressing cells) by single cell sorting, cells were validated for insertion efficiency, expression level, and functional effects. Finally successful clones were interrogated for their efficiency as tools for single molecule microscopy through measurements of effective resolution and cluster distribution.