Application of CRISPR/Cas9 to visualize defined genomic sequences in fixed chromosomes and nuclei

Takayoshi Ishii, Kiyotaka Nagaki, Andreas Houben

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Visualizing the spatio-temporal organization of the genome will improve our understanding of how chromatin structure and function are intertwined. Here, we describe a CRISPR/Cas9-based method to label in situ-specific DNA sequences. In contrast to classical fluorescence in situ hybridization (FISH), CRISPR-FISH does not require DNA denaturation and therefore permits better structural chromatin preservation. This method requires an assembled ribonucleoprotein (RNP) that consists of a target-specific CRISPR RNA (crRNA), a transactivating crRNA (tracrRNA), and recombinant dCas9 endonuclease protein. CRISPR-FISH can be used in fixed plant and animal cells and tissues to visualize specific DNA sequences together with protein and DNA replication sites. The broad range of adaptability of CRISPR-FISH to different temperatures and combinations of methods has the potential to advance the field of chromosome biology and cytogenomics.

Original languageEnglish
Title of host publicationCytogenomics
PublisherElsevier
Pages147-153
Number of pages7
ISBN (Electronic)9780128235799
DOIs
Publication statusPublished - Jan 1 2021

Keywords

  • CRISPR/Cas9
  • dCas9 endonuclease protein
  • DNA denaturation
  • Fluorescence in situ hybridization (FISH)
  • Ribonucleoprotein (RNP)
  • RNA-guided endonuclease-in situ labeling (CRISPR-FISH)
  • Target-specific CRISPR RNA (crRNA)
  • Transactivating crRNA (tracrRNA)

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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