(188cl) Single Molecule Investigation of TALE Protein's Genome-Wide Target Search in Live Cells

Transcription activator-like effector proteins (TALEs) are unique transcription factors that bind DNA in a nucleobase specific manner. TALEs have been widely used in genome editing applications because of their customizability and specificity. In vitro studies have revealed a novel two-state DNA search mechanism comprising of domain dependent sliding and hopping behavior. TALE assumes a superhelical structure around DNA, tracking the major groove. However, unlike DNA binding proteins belonging to the class of helical structures, TALEs undergo a rotationally decoupled search mechanism. These characteristics describe a novel DNA search mechanism of transcription factors that overcome the search speed-stability paradox. Yet the target search mechanism of TALE protein inside a highly dynamic and compact eukaryotic nucleus remains poorly understood. In this study, we elucidate TALE protein’s search mechanism in live mammalian cells by state-of the-art single particle tracking in euchromatin as well as heterochromatin. We report a single molecule imaging strategy based on Halotag fusion proteins which form a covalent bond with various synthetic dyes that have a reactive chloroalkane linker. This allows us to quantitatively measure specific and nonspecific dwell times on chromatin in single-live cells and determine in vivo target search time. We also compare the TALE DNA interrogation mechanism to CRISPR/Cas9 in the context of chromatin compaction to further understand the role of chromatin accessibility in genome editing applications and to establish design rules for construction of highly efficient and specific customizable DNA binding proteins.