Assaying break and nick-induced homologous recombination in mammalian cells using the DR-GFP reporter and Cas9 nucleases.
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.
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.
Lianne E M
LE
Vriend
Developmental Biology Program, Memorial Sloan-Kettering Cancer Center, New York, USA. Electronic address: m-jasin@mskcc.org.
Maria
M
Jasin
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.
Przemek M
PM
Krawczyk
D000818:Animals; D001483:Base Sequence; D064130:CRISPR-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D064113:CRISPR-Cas Systems; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D003850:Deoxyribonuclease I / Q000235:genetics / Q000378:metabolism*; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D017930:Genes, Reporter; D059765:Homologous Recombination; D006801:Humans; D008969:Molecular Sequence Data; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D064130:CRISPR-Associated Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D004720:Endonucleases; D003850:Deoxyribonuclease I
CRISPR/Cas; Cas9; DNA nicks; DNA repair; DR-GFP reporter; Homologous recombination; Single-strand breaks