Genome oligopaint via local denaturation fluorescence in situ hybridization

Mol Cell. 2021 Apr 1;81(7):1566-1577.e8. doi: 10.1016/j.molcel.2021.02.011. Epub 2021 Mar 2.

Abstract

Cas9 in complex with a programmable guide RNA targets specific double-stranded DNA for cleavage. By harnessing Cas9 as a programmable loader of superhelicase to genomic DNA, we report a physiological-temperature DNA fluorescence in situ hybridization (FISH) method termed genome oligopaint via local denaturation (GOLD) FISH. Instead of global denaturation as in conventional DNA FISH, loading a superhelicase at a Cas9-generated nick allows for local DNA denaturation, reducing nonspecific binding of probes and avoiding harsh treatments such as heat denaturation. GOLD FISH relies on Cas9 cleaving target DNA sequences and avoids the high nuclear background associated with other genome labeling methods that rely on Cas9 binding. The excellent signal brightness and specificity enable us to image nonrepetitive genomic DNA loci and analyze the conformational differences between active and inactive X chromosomes. Finally, GOLD FISH could be used for rapid identification of HER2 gene amplification in patient tissue.

Keywords: CRISPR-Cas9; DNA FISH; superhelicase.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • CRISPR-Associated Protein 9 / chemistry*
  • CRISPR-Cas Systems*
  • Cell Line
  • Female
  • Fibroblasts / chemistry
  • Fibroblasts / metabolism
  • Hot Temperature*
  • Humans
  • In Situ Hybridization, Fluorescence*
  • Nucleic Acid Denaturation*
  • RNA, Guide / chemistry*

Substances

  • RNA, Guide
  • CRISPR-Associated Protein 9