Dynamics of CRISPR-Cas9 genome interrogation in living cells.

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.

Single cell correlation fractal dimension of chromatin: a framework to interpret 3D single molecule super-resolution.

Chromatin is a major nuclear component, and it is an active matter of debate to understand its different levels of spatial organization, as well as its implication in gene regulation. Measurements of nuclear chromatin compaction were recently used to understand how DNA is folded inside the nucleus and to detect cellular dysfunctions such as cancer. Super-resolution imaging opens new possibilities to measure chromatin organization in situ. Here, we performed a direct measure of chromatin compaction at the single cell level. We used histone H2B, one of the 4 core histone proteins forming the nucleosome, as a chromatin density marker. Using photoactivation localization microscopy (PALM) and adaptive optics, we measured the three-dimensional distribution of H2B with nanometric resolution. We computed the distribution of distances between every two points of the chromatin structure, namely the Ripley K(r) distribution. We found that the K(r) distribution of H2B followed a power law, leading to a precise measurement of the correlation fractal dimension of chromatin of 2.7. Moreover, using photoactivable GFP fused to H2B, we observed dynamic evolution of chromatin sub-regions compaction. As a result, the correlation fractal dimension of chromatin reported here can be interpreted as a dynamically maintained non-equilibrium state.

Single-molecule tracking in live cells reveals distinct target-search strategies of transcription factors in the nucleus.

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.

Imaging transcription in living cells.

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.

Efficient optical trapping and visualization of silver nanoparticles.

We performed efficient optical trapping combined with sensitive optical detection of individual silver nanoparticles. The particles ranging in size from 20 to 275 nm in diameter were trapped in three dimensions using low laser power by minimizing spherical aberrations at the focus. The optical forces were quantified, and we found that the larger the particle, the stronger the optical force. The particles were imaged by an additional strongly scattered laser.