Journal
CYTOGENETIC AND GENOME RESEARCH
Volume 159, Issue 1, Pages 48-53Publisher
KARGER
DOI: 10.1159/000502600
Keywords
RGEN-ISL; CRISPR; Cas9; DNA replication; FISH; Immunostaining; Super-resolution microscopy
Categories
Funding
- Czech Science Foundation [17-14048S]
- ERASMUS+ student traineeship mobility program
- DFG [Ho1779/28-1]
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Visualizing the spatiotemporal organization of the genome will improve our understanding of how chromatin structure and function are intertwined. Here, we describe a further development of the CRISPR/Cas9-based RNA-guided endonuclease-in situ labeling (RGEN-ISL) method. RGEN-ISL allowed the differentiation between vertebrate-type (TTAGGG)(n) and Arabidopsis-type (TTTAGGG)(n) telomere repeats. Using maize as an example, we established a combination of RGEN-ISL, immunostaining, and EdU labeling to visualize in situ specific repeats, histone marks, and DNA replication sites, respectively. The effects of the non-denaturing RGEN-ISL and standard denaturing FISH on the chromatin structure were compared using super-resolution microscopy. 3D structured illumination microscopy revealed that denaturation and acetic acid fixation impaired and flattened the chromatin. The broad range of adaptability of RGEN-ISL to different combinations of methods has the potential to advance the field of chromosome biology.
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