Application of Tris-HCl Allows the Specific Labeling of Regularly Prepared Chromosomes by CRISPR-FISH

Visualizing the spatiotemporal organization of the genome will improve our understanding of how chromatin structure and function are intertwined. Here, we describe the further development of the RNA-guided endonuclease-in situ labeling (RGEN-ISL) method CRISPR-FISH. Using soybean and mouse chromosomes, we demonstrate that the treatment of conventionally fixed chromosomes (in ethanol or methanol:acetic acid) with 40 mM Tris-HCl (pH 9.0) for 30 minutes at 37 degrees C prior to CRISPR-FISH allows the application of this method for the detection of high-copy sequences. Wheat, rye, maize, andNicotiana benthamianawere used to confirm the applicability of the identified CRISPR-FISH conditions also in other species.