Characterization of Locus Specific Chromatin Structure and Dynamics using Correlative Conventional Super Resolution Crispr dCas9/Ms2 Imaging

Abstract

Chromatin undergoes dynamic rearrangements at the nucleosomal level that influences large scale genome organization. The characterization of both chromatin structure and dynamics has been key to study mechanisms of genome organization and its influence on gene regulation. Recently, CRISPR dCas9 based photo-activation localization microscopy (PALM) has been employed to resolve nucleosomal chromatin in fixed cells. However, the fast movement of DNA and long data acquisition time associated with PALM precludes the ability to obtain locus-specific structural and dynamic information in living cells. Furthermore, the highly variable mobility distribution of DNA and bound fluorescent dCas9 probes overlaps with the one of unbound dCas9, which makes it difficult to identify dCas9 molecules bound to chromatin based on their mobility alone. Here, we develop correlative conventional and PALM imaging to identify bound dCas9 molecules while correcting for the motion of a locus to obtain structural and dynamic information that would not be accessible with traditional PALM. in addition, we employ targeted photo-activation PALM via a digital mirror device to only photoactivate dCas9 probes in proximity to a locus. by taking advantage of the difference in interaction properties between bound and unbound probes, background localizations of non-interacting probes are dramatically reduced. We demonstrate with telomeres as a model system how the combination of these techniques makes it possible to obtain both structural and dynamic information that reveals novel quantitative information about chromatin structure, compaction, and condensation that would otherwise not be obtainable using existing imaging techniques.