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. 2022 Sep 14;32(11):969–981. doi: 10.1038/s41422-022-00712-z

Fig. 5. Simultaneous imaging of multiple CRISPR target loci tracks real-time dynamics of DNA DSB-induced chromosomal dissociation and subsequent intra- and interchromosomal rejoining in live cells.

Fig. 5

a Schematic diagram 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 h 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. c, d Time-lapse imaging and corresponding distance of DNA loci of pair 1 in b. Scale bar, 1 µm. e, f Time-lapse imaging and corresponding distance of DNA loci of pairs 2 and 3 in b. After separation and association of Chr3 fragments, pairs 2 and 3 gradually gathered as shown in e. Scale bar, 1 µm. g Schematic diagram of DSB-induced interchromosomal translocation between Chr3 and Chr13. The labeling strategy is similar to Fig. 4a. saCas9/sgRNA was delivered to produce DNA cutting between the labeled loci on Chr3 and SPACA7 gene on Chr13. h Representative images showing intrachromosomal dissociation and interchromosomal translocation between Chr3 and Chr13. Colored arrows indicate three DNA loci for tracking (green, PPP1R2; red, Chr3Rep; purple, Chr13Rep). The white box shows local enlargement. Time-lapse imaging started from 4 h post saCas9/sgRNA delivery. Scale bar, 1 µm. i Distance of DNA loci pairs shown in h. The red line shows the distance between Chr3Rep (red) and PPP1R2 (green) paired foci; the purple line shows the distance between Chr13Rep (purple) and PPP1R2 (green) paired foci.