Advances and challenges in CRISPR-based real-time imaging of dynamic genome organization

Abstract

Nuclear chromosome compaction is non-random and dynamic. The spatial distance among genomic elements instantly modulates transcription. Visualization of the genome organization in the cell nucleus is essential to understand nuclear function. In addition to cell type-dependent organization, high-resolution 3D imaging shows heterogeneous compaction of chromatin organization among the same cell type. Questions remain to be answered if these structural variations were the snapshots of dynamic organization at different time points and if they are functionally different. Live-cell imaging has provided unique insights into dynamic genome organization at short (milliseconds) and long (hours) time scales. The recent development of CRISPR-based imaging opened windows for studying dynamic chromatin organization in single cells in real time. Here we highlight these CRISPR-based imaging techniques and discuss their advances and challenges as a powerful live-cell imaging method that poses high potential to generate paradigm-shifting discoveries and reveal functional implications of dynamic chromatin organization.

Keywords: CRISPR; chromatin dynamics; chromosome conformation; genome organization; live-cell imaging; single-particle tracking.

FIGURE 1

A timeline of CRISPR-based imaging techniques. (Above the timeline) Various techniques which target repetitive sequences are illustrated moving from less SNR (left) to higher SNR (right). Cas variants consist of Cas proteins from orthogonal species fused to different fluorophores, which enable simultaneous multicolor imaging on multiple genomic loci (Chen et al., 2016). CRISPRainbow introduces ×2 stem loops, MS2, BoxB, and PP7, from bacteriophages into the sgRNA scaffold, which then bind with high affinity to their corresponding coat proteins with fluorescent tags, also enabling multicolor imaging of up to seven distinct repetitive sequences (Ma et al., 2016a). CRISPR LiveFISH attached a synthetic dye to the 5′ end of the sgRNA (Wang et al., 2019). CRISPR-Sirius builds on CRISPRainbow by expanding the MS2 and PP7 stem loops to ×8, increasing the fluorescent output and allowing tagging of lowly repetitive sequences (Ma et al., 2018). CRISPR-SunTag fused an array of ×24 GCN4 sites to the dCas9, which recruits the scFv antibody tethered to a green fluorescent protein. (Below the timeline) Various techniques which target non-repetitive sequences are illustrated. CRISPR-dual FRET MB engineered a gRNA which has a stem loop complementary to a set of molecular beacons containing a FRET pair. The donor and acceptor MB alone interact with their quenchers and are non-fluorescent (Mao et al., 2019). Once bound, the FRET pairs are in close proximity to one another, resulting in a bright fluorescent output. CasPLA is designed to detect single-nucleotide variations (SNVs) by using a pair of Cas9 complexes which target adjacent sequences (Zhang et al., 2018). The successful targeting leads to the ligation of linear probes bound to each sgRNA, which starts rolling circle amplification by the DNA polymerase. The generated long repetitive tail can be bound by fluorescent oligonucleotide probes for imaging. In the presence of a SNV, one of the Cas9 complexes will not bind which results in no fluorescent signal. Finally, Casilio amplifies signal by creating repeats within the sgRNA which are then bound by a fluorescently tagged RNA binding protein named Pumilio (Clow et al., 2022). Techniques listed on the timeline but not illustrated due to the space limit are: Cas9-GFP (Chen et al., 2013), CasFISH (Deng et al., 2015), CRISPR seqFISH (Takei et al., 2017), SNP-CLING (Maass et al., 2018), CRISPR-Tag (Chen et al., 2018), CRISPR Quantum Dot (Yang et al., 2020), and CRISPR-Sunspot (Sun et al., 2020).

FIGURE 2

Live-cell CRISPR imaging of genomic loci provides multiple forms of information about the locus and local chromatin domain. (A) Focusing on single cells can give temporal dynamics about the local chromatin region, using dual-colored techniques can show if the region is stably looped or unlooped by maintaining a small or large spatial distance between the foci, respectively. Alternatively, it informs if the loop is continuously modulating its structure by large fluctuations in the distance over time. (B) These techniques can indicate the local chromatin compaction by tracking the trajectories of the fluorescent foci over time. Highly packed chromatin (e.g., chromatin in early G1 phase) is generally more stiff thus minimizing locus movement compared to loosely packed chromatin (e.g., chromatin in late G1 or early S phase). (C) Although introducing multiple labels per chromosomal domain allows the visualization of the chromosome territory (chromosome painting), using a multicolor labeling strategy will provide the structural information in detail (multicolor labeling).