CRISPR/Cas9-Based RGEN-ISL Allows the Simultaneous and Specific Visualization of Proteins, DNA Repeats, and Sites of DNA Replication

Němečková, A., Wäsch, Ch., Schubert, V., Ishii, T., Hřibová, E., Houben, A.

Klíčová slova: CRISPR/Cas9; DNA replication; FISH; Immunostaining; RGEN-ISL; Super-resolution microscopy
Abstrakt: 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.
DOI: 10.1159/000502600
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