Journal
NEW PHYTOLOGIST
Volume 222, Issue 3, Pages 1652-1661Publisher
WILEY
DOI: 10.1111/nph.15720
Keywords
centromere; CRISPR; Cas9; FISH; immunostaining; in situ labelling; telomere
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Funding
- CSIRO from the BMGF (USA)
- Deutsche Forschungsgemeinschaft DFG [Ho1779/28-1]
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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.
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