RNA‐guided endonuclease – in situ labelling (RGEN‐ISL): a fast CRISPR/Cas9‐based method to label genomic sequences in various species

Visualising the spatio‐temporal organisation of the genome will improve our understanding of how chromatin structure and function are intertwined. We developed a tool to visualise defined genomic sequences in fixed nuclei and chromosomes based on a two‐part guide RNA with a recombinant Cas9 endonuclease complex. This method does not require any special construct or transformation method. In contrast to classical fluorescence in situ hybridiaztion, RGEN‐ISL (RNA‐guided endonuclease – in situ labelling) does not require DNA denaturation, and therefore permits a better structural chromatin preservation. The application of differentially labelled trans‐activating crRNAs allows the multiplexing of RGEN‐ISL. Moreover, this technique is combinable with immunohistochemistry. Real‐time visualisation of the CRISPR/Cas9‐mediated DNA labelling process revealed the kinetics of the reaction. The broad range of adaptability of RGEN‐ISL to different temperatures and combinations of methods has the potential to advance the field of chromosome biology.