Application of Tris-HCl Allows the Specific Labeling of Regularly Prepared Chromosomes by CRISPR-FISH
Bhanu Prakash Potlapalli, Veit Schubert, Janina Metje‐Sprink, Thomas Liehr, Andreas Houben
Abstract
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°C prior to CRISPR-FISH allows the application of this method for the detection of high-copy sequences. Wheat, rye, maize, and Nicotiana benthamiana were used to confirm the applicability of the identified CRISPR-FISH conditions also in other species.