Development of a CRISPR-imaging toolset for imaging of DNA and RNA in structurally preserved chromatin suitable for super-resolution microscopy and electron microscopy and its application to study the substructure of holo- and monocentromeres

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Temporal and spatial organization of chromosomes and nuclei is increasingly recognized as being important for regulating functions such as gene expression, DNA replication and repair, as well as proper segregation of the genetic material during cell division. We aim to harness the CRISPR-Cas9 system to develop a CRISPR-imaging toolset for imaging of DNA and RNA in structurally preserved chromatin suitable for super-resolution microscopy and electron microscopy. We intend to develop CRISPR-FISH methods with an improved fluorescence reporter suitable to detect single and low copy DNA in non-denatured, native chromatin; for the detection of specific DNA sequences by electron microscopy at high resolution and to fluorescence-label specific RNA transcripts. These pioneering methods will be used to analyze the substructure of holo- and monocentromeres at different stages of the cell cycle by electron and super-resolution microscopy.