Assaying break and nick-induced homologous recombination in mammalian cells using the DR-GFP reporter and Cas9 nucleases.
Methods Enzymol
; 546: 175-91, 2014.
Article
em En
| MEDLINE
| ID: mdl-25398341
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.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Temas:
Geral
Base de dados:
MEDLINE
Assunto principal:
Genes Reporter
/
Desoxirribonuclease I
/
Reparo do DNA
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Quebras de DNA de Cadeia Dupla
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Recombinação Homóloga
/
Proteínas Associadas a CRISPR
Limite:
Animals
/
Humans
Idioma:
En
Revista:
Methods Enzymol
Ano de publicação:
2014
Tipo de documento:
Article
País de afiliação:
Holanda