CtIP fusion to Cas9 enhances transgene integration by homology-dependent repair.
Nat Commun
; 9(1): 1133, 2018 03 19.
Article
em En
| MEDLINE
| ID: mdl-29556040
ABSTRACT
In genome editing with CRISPR-Cas9, transgene integration often remains challenging. Here, we present an approach for increasing the efficiency of transgene integration by homology-dependent repair (HDR). CtIP, a key protein in early steps of homologous recombination, is fused to Cas9 and stimulates transgene integration by HDR at the human AAVS1 safe harbor locus. A minimal N-terminal fragment of CtIP, designated HE for HDR enhancer, is sufficient to stimulate HDR and this depends on CDK phosphorylation sites and the multimerization domain essential for CtIP activity in homologous recombination. HDR stimulation by Cas9-HE, however, depends on the guide RNA used, a limitation that may be overcome by testing multiple guides to the locus of interest. The Cas9-HE fusion is simple to use and allows obtaining twofold or more efficient transgene integration than that with Cas9 in several experimental systems, including human cell lines, iPS cells, and rat zygotes.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Proteínas Nucleares
/
Proteínas de Transporte
/
Reparo de DNA por Recombinação
Limite:
Animals
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Female
/
Humans
Idioma:
En
Ano de publicação:
2018
Tipo de documento:
Article