Progress and Challenges for Live-Cell Imaging of Genomic Loci using CRISPR-Based Platforms

Abstract

Chromatin conformation, localization, and dynamics are crucial regulators of cellular behaviors. Although fluorescence in situ hybridization-based techniques have been widely utilized for investigating chromatin architectures in healthy and diseased states, the requirement for cell fixation precludes the comprehensive dynamic analysis necessary to fully understand chromatin activities. This has spurred the development and application of a variety of imaging methodologies for visualizing single chromosomal loci in the native cellular context. In this review, we describe currently-available approaches for imaging single genomic loci in cells, with special focus on clustered regularly interspaced short palindromic repeats ( CRISPR )-based imaging approaches. In addition, we discuss some of the challenges that limit the application of CRISPR-based genomic imaging approaches, and potential solutions to address these challenges. We anticipate that, with continued refinement of CRISPR-based imaging techniques, significant understanding can be gained to help decipher chromatin activities and their relevance to cellular physiology and pathogenesis.

Applications of CRISPR/Cas9 tools in deciphering the mechanisms of HIV-1 persistence

“…Indeed, much of our understanding of the impact of nuclear topography to HIV-1 gene regulation relies on immuno-three-dimensional fluorescent in situ hybridization (immune-FISH). This technique uses multiple tagged probes to target a nucleotidic sequence of interest resulting in visualizable discrete bright spot indicative of a single locus [53]. Because the cells need to be fixed, FISH provides high spatial but static information [53].…”

“…This technique uses multiple tagged probes to target a nucleotidic sequence of interest resulting in visualizable discrete bright spot indicative of a single locus [53]. Because the cells need to be fixed, FISH provides high spatial but static information [53]. On the contrary, CRISPR/dCas9-based techniques allow for noninvasive live imaging of genomic loci [53].…”

“…Because the cells need to be fixed, FISH provides high spatial but static information [53]. On the contrary, CRISPR/dCas9-based techniques allow for noninvasive live imaging of genomic loci [53]. A wide variety of CRISPR-based imaging methodologies have been developed in the recent years, using dCas9 fused with classical fluorochromes, more stable and smaller organic dyes or specific nanoparticles [53].…”

“…On the contrary, CRISPR/dCas9-based techniques allow for noninvasive live imaging of genomic loci [53]. A wide variety of CRISPR-based imaging methodologies have been developed in the recent years, using dCas9 fused with classical fluorochromes, more stable and smaller organic dyes or specific nanoparticles [53]. This latter approach was recently reported by the team of Xian-En Zhang to live visualizing single-copy of integrated proviral HIV-1 DNA in latently infected cells [54 ].…”

“…This latter approach was recently reported by the team of Xian-En Zhang to live visualizing single-copy of integrated proviral HIV-1 DNA in latently infected cells [54 ]. Indeed, the team used a dCas9 fused with luminescent nanoparticles termed quantum dots, that are brighter and have a higher photostability than organic dyes and fluorochromes, and that allow single-molecule imaging [53]. More specifically, a pair of dCas9 fused to two differently colored quantum dots was co-targeted to the HIV-1 promoter region with optimized sgRNA and allowed to successfully image HIV-1 proviral DNA in latently infected cells (Figure 2c) [54].…”