@article {47160, title = {Dynamics of CRISPR-Cas9 genome interrogation in living cells.}, journal = {Science (New York, N.Y.)}, volume = {350}, year = {2015}, month = {2015 Nov 13}, pages = {823-6}, abstract = {

The RNA-guided CRISPR-associated protein Cas9 is used for genome editing, transcriptional modulation, and live-cell imaging. Cas9-guide RNA complexes recognize and cleave double-stranded DNA sequences on the basis of 20-nucleotide RNA-DNA complementarity, but the mechanism of target searching in mammalian cells is unknown. Here, we use single-particle tracking to visualize diffusion and chromatin binding of Cas9 in living cells. We show that three-dimensional diffusion dominates Cas9 searching in vivo, and off-target binding events are, on average, short-lived (\<1 second). Searching is dependent on the local chromatin environment, with less sampling and slower movement within heterochromatin. These results reveal how the bacterial Cas9 protein interrogates mammalian genomes and navigates eukaryotic chromatin structure.

}, issn = {1095-9203}, doi = {10.1126/science.aac6572}, author = {Knight, Spencer C and Xie, Liangqi and Deng, Wulan and Guglielmi, Benjamin and Witkowsky, Lea B and Bosanac, Lana and Zhang, Elisa T and El Beheiry, Mohamed and Masson, Jean-Baptiste and Dahan, Maxime and Liu, Zhe and Doudna, Jennifer A and Tjian, Robert} } @article {45338, title = {Single-molecule tracking in live cells reveals distinct target-search strategies of transcription factors in the nucleus.}, journal = {eLife}, year = {2014}, month = {2014 Jun 12}, pages = {e02230}, abstract = {

Gene regulation relies on transcription factors (TFs) exploring the nucleus searching their targets. So far, most studies have focused on how fast TFs diffuse, underestimating the role of nuclear architecture. We implemented a single-molecule tracking assay to determine TFs dynamics. We found that c-Myc is a global explorer of the nucleus. In contrast, the positive transcription elongation factor P-TEFb is a local explorer that oversamples its environment. Consequently, each c-Myc molecule is equally available for all nuclear sites while P-TEFb reaches its targets in a position-dependent manner. Our observations are consistent with a model in which the exploration geometry of TFs is restrained by their interactions with nuclear structures and not by exclusion. The geometry-controlled kinetics of TFs target-search illustrates the influence of nuclear architecture on gene regulation, and has strong implications on how proteins react in the nucleus and how their function can be regulated in space and time.

}, issn = {2050-084X}, doi = {10.7554/eLife.02230}, author = {Izeddin, Ignacio and R{\'e}camier, Vincent and Bosanac, Lana and Cisse, Ibrahim I and Boudarene, Lydia and Dugast-Darzacq, Claire and Proux, Florence and B{\'e}nichou, Olivier and Voituriez, Rapha{\"e}l and Bensaude, Olivier and Dahan, Maxime and Darzacq, Xavier} } @article {2390, title = {Imaging transcription in living cells.}, journal = {Annual Review of Biophysics}, volume = {38}, year = {2009}, month = {2009 Feb 3}, pages = {173-96}, abstract = {

The advent of new technologies for the imaging of living cells has made it possible to determine the properties of transcription, the kinetics of polymerase movement, the association of transcription factors, and the progression of the polymerase on the gene. We report here the current state of the field and the progress necessary to achieve a more complete understanding of the various steps in transcription. Our Consortium is dedicated to developing and implementing the technology to further this understanding.

}, keywords = {Computer-Assisted, Fluorescence, Gene Expression Profiling, Image Interpretation, Microscopy, Transcription Factors, Transcriptional Activation}, issn = {1936-122X}, doi = {10.1073/pnas.1100640108}, author = {Darzacq, Xavier and Yao, Jie and Larson, Daniel R and Causse, Sebastien Z and Bosanac, Lana and de Turris, Valeria and Ruda, Vera M and Lionnet, Timoth{\'e}e and Zenklusen, Daniel and Guglielmi, Benjamin and Tjian, Robert and Singer, Robert H} }