Zooming in on chromosome dynamics

Abstract

Until recently, our understanding of chromosome organization in higher eukaryotic cells has been based on analyses of large-scale, low-resolution changes in chromosomes structure. More recently, CRISPR-Cas9 technologies have allowed us to "zoom in" and visualize specific chromosome regions in live cells so that we can begin to examine in detail the dynamics of chromosome organization in individual cells. In this review, we discuss traditional methods of chromosome locus visualization and look at how CRISPR-Cas9 gene-targeting methodologies have helped improve their application. We also describe recent developments of the CRISPR-Cas9 technology that enable visualization of specific chromosome regions without the requirement for complex genetic manipulation.

FISH Going Meso-Scale: A Microscopic Search for Chromatin Domains

“…Tracing of individual chromatin contact domains in vivo would allow more precise analysis of chromatin fiber behavior, domain persistence time, stochastic and specific interactions, and other parameters, which are crucial for understanding the principles behind chromatin domain formation. Attaining this goal is tightly linked to the development of probes for in vivo labeling of genomic loci (both artificially inserted and endogenous), generally based on the operator-repressor methods (LacO/LacI, TetO/TetR), ANCHOR/ParB system, transcription activator-like effectors (TALEs) or clustered regularly interspaced short palindromic repeats (CRISPR)/nuclease-deactivated CRISPR-associated protein 9 (dCas9) technology (reviewed by Eykelenboom and Tanaka, 2020 ). CRISPR/dCas9 technology utilizes single guide RNAs (sgRNAs) to target complementary DNA locus and recruit dCas9 fused with fluorescent protein toward the DNA/RNA duplex ( Chen et al, 2013 ).…”

Assessing the polymer coil-globule state from the very first spectral modes

“…In order to get access to the first M modes, indeed, it is in principle sufficient to record configurations of M distinguishable, equally spaced monomers covering the whole chain, either by following their dynamics or by averaging over a collection of identical polymers to ensure enough statistics. The design of multicolor fluorescent imaging of DNA sites with high spatial and temporal resolution now makes it possible to record the trajectories and relative positions of multiple loci simultaneously [21][22][23]. If the DCT-transform of a set of M -point configurations contain the equivalent of the first modes for the whole chain as we predict, this yields a new experimental approach for the determination of the state of single polymers and biopolymers.…”

Assessing the polymer coil-globule state from the very first spectral modes

“…In order to get access to the first M modes, indeed, it is in principle sufficient to record configurations of M distinguishable, equally spaced monomers covering the whole chain, either by following their dynamics or by averaging over a collection of identical polymers to ensure enough statistics. The design of multicolor fluorescent imaging of DNA sites with high spatial and temporal resolution now makes it possible to record the trajectories and relative positions of multiple loci simultaneously [21][22][23]. If the DCT-transform of a set of M -point configurations contain the equivalent of the first modes for the whole chain as we predict, this yields a new experimental approach for the determination of the state of single polymers and biopolymers.…”