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CRISPR FISHer Enables High-sensitivity Imaging of Nonrepetitive DNA in Living Cells Through Phase Separation-mediated Signal Amplification

27, 2022

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Nuclear DNA, including chromosomal and extrachromosomal DNA elements, is highly dynamic and spatiotemporally organized to control the fundamental cellular processes and beyond. Genome instability or chromosomal structural variations could induce DNA damage and repair, sometimes generating extrachromosomal DNAs. In addition, some invaders, such as viruses, could infect the cell and deliver its genome into the nucleus, resulting in cellular dysfunction and disease development. Therefore, visualizing the spatial distribution and dynamics of nuclear DNA, including chromosomal and extrachromosomal DNA elements, is pivotal to understanding their biological functions. However, visualizing any specified endogenous single-copy locus, or invading DNA locus of interest, is still a crucial challenge in living cells.

Previous studies have achieved genomic imaging by integrating large arrays of artificial DNA sequences, such as LacO, which depends on tedious genome engineering for each locus of interest and may disturb the target loci and induce unpredicted side effects by inserting exogenous sequences. The recent promising CRISPR-mediated live-cell DNA imaging requires multiple repetitive elements or an array of sgRNAs tiled along the target locus. These requirements preclude the widespread use of CRISPR imaging. For example, few tandem DNA repeats for a specific locus exist in the human genome; the imaging strategy using sgRNAs tiling has trouble with signal-to-background, making it challenging to visualize the native nonrepeated DNA sequences.

The groups of Drs. Chun-Qing Song and En-Zhi Shen have developed a novel DNA live imaging technique CRISPR FISHer, which achieves the high-sensitivity imaging of nonrepetitive DNA in living cells through phase separation-mediated signal amplification. The research was published in Cell Research in Sept. 2022 and selected as the cover story.

1) CRISPR FISHer achieves unprecedented brightness and sensitivity at CRISPR targeting DNA loci. i.e., the signal-to-noise ratio in CRISPR FISHer at repetitive telomere loci is up to 246-fold, while that in traditional CRISPR labeling strategies is less than 2 (Fig. 1).

Fig. 1 CRISPR FISHer enables robust repetitive genomic loci tracking with enhanced signal-to-background. (a) Comparison of foldon-GFP-PCP, PCP-GFP, and dCas9-EGFP labeling of telomere loci in U2OS cells. sgGal4 is used as the negative control. (b) Comparison of signal-to-background (S/B) ratio of labeled telomere loci using foldon-GFP-PCP, PCP-GFP, and dCas9-EGFP.

2) With a single sgRNA, CRISPR FISHer enables the detection and real-time live imaging of endogenous nonrepetitive DNA sequences in living cells (Fig. 2).

Fig. 2 CRISPR FISHer allows for visualization of the endogenous nonrepetitive genomic region. (a) Representative images for PPP1R2 gene signal labeled by PCP-GFP or foldon-GFP-PCP. Scale bar, 5 µm. (b) Representative PPP1R2 loci distribution in serial Z-sections for (a). (c and d) Comparison of foldon-GFP-PCP and PCP-GFP labeling of single-copy gene PPP1R2 (green); repetitive Chr3Rep loci (red, internal control). The dotted lines (left) mark area for producing line scans (right). Scale bar, 5 µm.

3) In one application of visualizing one of the fundamental nuclear events, by labeling two broken ends of chromosomal fragments, we, for the first time, observed the consecutive process of DNA double-strand break (DSB) induced by CRISPR and subsequent non-homologous ending joining (NHEJ) repair within the same chromosomes in a single living cell (Fig. 3). In addition, we also observed the consecutive process of chromosomal breakage and consequent translocation in a single live cell.

Fig. 3 Simultaneous imaging of multiple CRISPR target loci tracks dynamics of DNA DSB-induced chromosomal dissociation and subsequent intrachromosomal rejoining in live cells. (a) Schematic of intrachromosomal separation and rejoining through labeling both sides of the cutting site. Chr3Rep and PPP1R2 gene on Chr3 were labeled by CRISPR Sirius and CRISPR FISHer in U2OS cells. SaCas9/sgRNA was delivered by nucleofection (16 hours after delivering DNA loci labeling systems) for inducing DSB between the two labeled loci. (b) Representative fluorescent images of DSB-induced intrachromosomal dissociation and rejoining in a single cell. White boxes show DNA loci pairs. Scale bar, 5 µm.

4) Beyond the chromosomal DNA, CRISPR FISHer is able to detect and track the real-time movement of extrachromosomal DNA, including native extrachromosomal circular DNA (eccDNA, Fig. 4) and invading DNAs HBV and AAV. We found that the non-integrated extrachromosomal DNAs (native eccDNA, linear eccDNA, and AAV DNA) exhibited extremely different dynamic properties from the chromosomal DNA.

Fig. 4 CRISPR FISHer detection and real-time visualization of native extrachromosomal eccDNAs in live cells. (a) The schematic strategy of the eccDNA labeling with CRISPR FISHer. sgRNA target sites located at junction regions of eccDNAs. (b and c) Representative images showing labeled eccDNAs by CRISPR FISHer in HepG2 cells (b) and the number of eccDNA foci (c). (d) Representative XYZ-t trajectories for eccBEND3 and Chr3 loci during a 5-min period. (e) Comparison of tracking length of eccBEND3 and Chr3 in HepG2 cells.

Therefore, CRISPR FISHer broadens the scope of DNA live imaging, including both endogenous and exogenous DNAs. It not only provides scientists a platform to real-time visualize any specified DNA molecules but also inspires the study of fundamental nuclear events by labeling genes of interest. Furthermore, CRISPR FISHer opens a door to biomedical diagnosis and related mechanistic study by detecting and tracking invading DNAs.