Visualization of Genomic Loci in Living Cells with a Fluorescent CRISPR/Cas9 System

Methods Mol Biol. 2016:1411:407-17. doi: 10.1007/978-1-4939-3530-7_25.

Abstract

The discovery that the RNA guided bacterial endonuclease Cas9 can be harnessed to target and manipulate user-defined genomic sequences has greatly influenced the field of genome engineering. Interestingly, a catalytically dead Cas9 (dCas9) can be employed as a targeted DNA-binding platform to alter gene expression. By fusing this dCas9 to eGFP, we and others could show that the CRISPR/Cas9 system can be further expanded to label and trace genomic loci in living cells. We demonstrated that by exchanging the sgRNA, dCas9-eGFP could be specifically directed to various heterochromatic sequences within the nucleus. Here, we provide a basic protocol for this versatile tool and describe how to verify new dCas9-eGFP targets.

Keywords: CRISPR/Cas9; In vivo labeling; Repetitive sequences; sgRNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CRISPR-Cas Systems*
  • Cells, Cultured
  • Chromosome Mapping* / methods
  • Cloning, Molecular
  • Gene Expression
  • Gene Targeting
  • Genes, Reporter
  • Genetic Loci*
  • Genome*
  • In Situ Hybridization, Fluorescence
  • Mice
  • Microscopy, Fluorescence
  • RNA, Guide, CRISPR-Cas Systems
  • Repetitive Sequences, Nucleic Acid
  • Transfection

Substances

  • RNA, Guide, CRISPR-Cas Systems