Single molecule investigation of transcription activator-like effector search dynamics

Recent advances in genetic engineering hold great potential to profoundly change the treatment of human disease. Precise manipulation of genetic material allows for the creation of new disease models and the rapid translation of new therapies into the clinic. Several classes of programmable nucleases allowing for precise and targeted genomic edits are central to the rising popularity, flexibility, and accessibility of gene engineering. Transcription activator-like effectors (TALEs) are one such class that form a powerful gene-editing platform when fused to nuclease domains, thereby yielding TALEN systems. Despite pervasive use of TALENs for editing crops, small animals, eukaryotic stem cells, and human T-cells, remarkably little is known about the molecular mechanisms used to locate and bind their DNA target sites. This work describes the application of single molecule fluorescence imaging to the study the TALE search process along specific and non-specific DNA templates. Our work provides a molecular-level picture of the dynamics of TALE-DNA interactions, and our results have revealed an apparently unique search mechanism for DNA binding proteins. We directly observe TALEs diffusing along non-specific DNA in one-dimension. Our results show that TALE diffusion occurs in a directionally unbiased and thermally driven manner along double surface tethered and extended DNA templates (Chapter 2). Interestingly, we observe significant intra-trajectory heterogeneity for diffusion of full-length TALE proteins. We further isolate and study the single molecule dynamics of TALE truncation mutants containing only the N-terminal region (NTR), and these results reveal the importance of the NTR for nucleating non-specific binding. We find that the TALE NTR alone is capable of short, rapid non-specific search. Furthermore, we study the diffusion of a series of TALEs with variable size central repeat domains (CRDs). Taken together with insights from NTR dynamics and the heterogeneity of full-length TALE diffusion, we propose a two-state search mechanism for TALEs that is comprised of rapid search and interspersed periods of local sequence checking along DNA templates. We further expand our characterization of TALE search by determining the impact of solution conditions, ionic strength, probe size, and the role of hydrodynamic flow on TALE dynamics (Chapter 3). Using this combination of single molecule experiments, we find that TALE diffusion does not fit the traditional definitions of binary classification of DNA-binding protein search, which have been characterized as protein hopping or sliding along DNA templates. Instead, our results suggest a mechanism wherein TALEs encircle DNA templates during search, but form only transient contacts with the DNA backbone. Furthermore, the non-specific search trajectory of TALEs is rotationally decoupled, in contrast to a broad class of other DNA binding proteins including DNA repair proteins and transcription factors. We further utilize a combination of bulk fluorescence anisotropy measurements and single molecule experiments to characterize the effects of divalent cations on TALE binding (Chapter 4). Our results show that TALE specificity is significantly enhanced in the presence of certain divalent cations, which can be attributed to a decrease in non-specific binding affinity. Finally, we generate long DNA templates with TALE target binding arrays at precise locations, and we directly visualize specific binding and localization of TALEs to their respective target sites following 1-D search. Taken together, our results have elucidated the fundamental search mechanism of TALE proteins along DNA templates