Assaying break and nick-induced homologous recombination in mammalian cells using the DR-GFP reporter and Cas9 nucleases

Methods Enzymol. 2014;546:175-91. doi: 10.1016/B978-0-12-801185-0.00009-X.

Abstract

Thousands of DNA breaks occur daily in mammalian cells, including potentially tumorigenic double-strand breaks (DSBs) and less dangerous but vastly more abundant single-strand breaks (SSBs). The majority of SSBs are quickly repaired, but some can be converted to DSBs, posing a threat to the integrity of the genome. Although SSBs are usually repaired by dedicated pathways, they can also trigger homologous recombination (HR), an error-free pathway generally associated with DSB repair. While HR-mediated DSB repair has been extensively studied, the mechanisms of HR-mediated SSB repair are less clear. This chapter describes a protocol to investigate SSB-induced HR in mammalian cells employing the DR-GFP reporter, which has been widely used in DSB repair studies, together with an adapted bacterial CRISPR/Cas system.

Keywords: CRISPR/Cas; Cas9; DNA nicks; DNA repair; DR-GFP reporter; Homologous recombination; Single-strand breaks.

MeSH terms

  • Animals
  • Base Sequence
  • CRISPR-Associated Proteins / genetics
  • CRISPR-Associated Proteins / metabolism*
  • CRISPR-Cas Systems
  • Cell Line
  • Cloning, Molecular / methods
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • DNA Breaks, Double-Stranded*
  • DNA Repair*
  • Deoxyribonuclease I / genetics
  • Deoxyribonuclease I / metabolism*
  • Endonucleases / genetics
  • Endonucleases / metabolism
  • Flow Cytometry / methods
  • Genes, Reporter*
  • Homologous Recombination*
  • Humans
  • Molecular Sequence Data
  • RNA, Guide / genetics
  • RNA, Guide / metabolism
  • Transfection

Substances

  • CRISPR-Associated Proteins
  • RNA, Guide
  • Endonucleases
  • Deoxyribonuclease I