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RNA ‐guided endonuclease – in situ labelling ( RGEN ‐ ISL ): a fast CRISPR /Cas9‐based method to label genomic sequences in various species
Author(s) -
Ishii Takayoshi,
Schubert Veit,
Khosravi Solmaz,
Dreissig Steven,
MetjeSprink Janina,
Sprink Thorben,
Fuchs Jörg,
Meister Armin,
Houben Andreas
Publication year - 2019
Publication title -
new phytologist
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.742
H-Index - 244
eISSN - 1469-8137
pISSN - 0028-646X
DOI - 10.1111/nph.15720
Subject(s) - chromatin , endonuclease , cas9 , rna , computational biology , biology , oligonucleotide , dna , crispr , microbiology and biotechnology , genetics , gene
Summary 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 cr RNA s 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.