Characterization Of Dcas9 Interaction Kinetics And Local Chromatin Structure In Live Human Cells Using Palm Superresolution Microscopy

Gene regulation involves the dynamic rearrangement of chromatin but mapping both the spatial organization of chromatin and its dynamics remains a challenge. Many important structural conformations are too small to be resolved via conventional fluorescence microscopy and the long acquisition time of super-resolution PALM imaging makes mapping the organization of mobile chromatin difficult. Recently, CRISPR-dCas9 has been employed for live cell DNA imaging because of the ability to add multiple gRNAs that target different sequences at once. PALM is able to track individual dCas9 molecules, which can either bind to their target site, scan DNA searching for their target site, or freely diffuse. Determining the mobility state of individual Cas9 molecules is critical to mapping the spatial organization of dCas9 molecules bound to mobile chromatin. The diffusion coefficient distributions of the bound and searching dCas9 populations overlap due to variability in DNA mobility, making it difficult to determine the mobility state of Cas9 by only analyzing mobility transitions in a single trace. Here we employ correlative conventional fluorescence and live cell PALM imaging to quantitatively map chromatin organization while tracking its dynamics. By tagging both the dCas9 and the gRNA with conventional and photoactivatable probes, we assign molecules to a specific cluster and separate the bound population from the scanning population. From this, we can account for changes in DNA mobility while measuring locus specific dCas9 residence times in the bound state and more accurately calculate interaction kinetics, which provide insights into local chromatin accessibility. This correlative conventional and PALM approach therefore improves the ability to exclusively analyze the mobility and spatial distribution of locus-specific target bound chromatin with single molecule precision.