Format

Send to

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

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

Author information

1
Developmental Biology Program, Memorial Sloan-Kettering Cancer Center, New York, USA; Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
2
Developmental Biology Program, Memorial Sloan-Kettering Cancer Center, New York, USA. Electronic address: m-jasin@mskcc.org.
3
Developmental Biology Program, Memorial Sloan-Kettering Cancer Center, New York, USA; Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. Electronic address: p.krawczyk@amc.uva.nl.

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

[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Elsevier Science Icon for PubMed Central
Loading ...
Support Center