RNA‐guided endonuclease – in situ labelling (RGEN‐ISL): a fast CRISPR/Cas9‐based method to label genomic sequences in various species
Citations Over TimeTop 15% of 2019 papers
Abstract
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.
Related Papers
- → Effective PEI-mediated delivery of CRISPR-Cas9 complex for targeted gene therapy(2018)93 cited
- → Non-viral and viral delivery systems for CRISPR-Cas9 technology in the biomedical field(2017)52 cited
- → Cas9 Cuts and Consequences; Detecting, Predicting, and Mitigating CRISPR/Cas9 On‐ and Off‐Target Damage(2020)20 cited
- → [Research progress of CRISPR-Cas9 system for gene therapy].(2016)6 cited
- → Prediction of off-target effects of the CRISPR/Cas9 system for design of sgRNA(2020)